Biochemistry

2019
 Adamson University
SY 2018 – 2019 Carbohydrates 1 May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Nerona & Gungon

• Furanose, because they resemble the five membered ring

Outline of the Lecture compound furan.
I. Carbohydrates
a. Monosaccharides
b. Disaccharides
c. Oligosaccharides
d. Polysaccharides
II. Carbohydrate Digestion
III. Carbohydrates Diseases

Carbohydrates

• Most abundant biological molecule on earth Fischer Projection

The 2-dimensional representation of a 3-dimensional
• Produced from CO2 fixation during Photosynthesis
organic molecule. Were originally proposed for the
• Minimum of 3 carbon
depiction of carbohydrates.
• Used as energy storage [Starch/sugar]
• Some insoluble carbohydrates polymers can serve as
structural or protective support. [plant/animal cell wall
and animal connective tissues]
• Can also participate in cell recognition

Anomeric Carbon: carbonyl carbon which became the chiral

center during cyclization of sugars
• Alpha: -OH in the anomeric C is down
• Beta: -OH in the anomeric C is up
• “ABBA” = above-beta, below-alpha
Chiral Center: carbon with 4 non-identical substituents Examples:
attached to it; determines chirality

Monosaccharides
• Simple sugars, cannot be broken further down
• Empirical formula = (CH2O)n
• Can come in the form of Ketose or Aldose [Containing
Ketone and Aldose functional group respectively]
• All monosaccharides are reducing sugars
• Important monosaccharides: glucose, mannose, fructose,
and galactose
• General Formula: C6H12O6
• Aldoses with 3C or more and ketoses with 4C or more are
chiral
• Commonly Found in the D orientation in nature

Stereochemistry
• Enantiomers = mirror images [L or D].
• Diastereomers = Pairs of isomers that have opposite
configurations at one or more chiral centers but are NOT
mirror images.
• Epimers = Pairs that differ in configuration at only one
chiral center.

Haworth Projection
Used to show Cyclic Conformation of a Carbohydrate
• Pyranose, because they resemble the six membered ring
compound pyran.

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Aldoses

Derivatives
Changing of a -OH group with another Functional Group

Amino Sugar

Deoxy sugar

Sugar Phosphates

Disaccharides
Two monosaccharides compounded together by glycosidic
bond

Glycosidic Bond
• A glycosidic bond is formed between the hemiacetal or
hemiketal group of a saccharide (or a molecule derived
from a saccharide) and the hydroxyl group of some
Ketoses
compound such as an alcohol.
• Covalent bond formed between monomeric sugars in a
carbohydrate chain

• 2 types:
 O-glycosidic bond - -OH and aldehyde
 N-glycosidic bond - -OH and amine

Important disaccharides: ▪
• Sucrose: glucose + fructose (α-1,2-linkage), nonreducing
sugar, table sugar
2
• Maltose: glucose + glucose (α-1,4-linkage), reducing sugar,
malt sugar
• Lactose: glucose + galactose (α-1,4-linkage), reducing sugar,
milk sugar
Reducing Sugars
• All monosaccharides are Reducing sugars
• Disaccharides consist of two monosaccharides and may be
either reducing or nonreducing.
• The reducing end of a disaccharide is the monosaccharide
with a free anomeric carbon that is not involved in a
glycosidic bond and is thus capable of converting to the
open-chain form.
*Fehlings and Benedicts Test can be used to determine
reducing sugars, but most disaccharides react to these tests
slowly
Common disaccharides:
3
Oligosaccharides: 3-12 monosaccharides linked together
Polysaccharides
• Multiple oligosaccharide units
• Functions: for energy storage (starch and glycogen) and for
maintenance of structural integrity (cellulose and chitin).
• Important polysaccharides:
 Glycogen: - α (1→4) glycosidic bond (linear chain) and α
(1→6) glycosidic bond (branched chain)
 Chitin: β (1→4) glycosidic bond
Important Polysaccharides:
 Cellulose
• Chief constituent of plant cell walls (structural)
• Insoluble
• β-Dglucopyranose units linked by β (1-4) bonds (long,
straight chains strengthened by crosslinking hydrogen
bonds)
• Mammals lack cellulase (enzyme that hydrolyze β 1-4
bonds), and thus cannot digest cellulose
• Important source of “bulk” in the diet, and the major
component of dietary fiber (no nutritional value)
• There is some bacterial metabolism of cellulose in the
human colon.
 Starch
• Is a homopolymer of glucose forming a α-glucosidic chain,
called a glucosan or glucan
• “Vehicles for storage of glucose”
2 Main Constituents
1. Amylose (13%- 20%)
• Non-branching, linear polymer of glucose
• Residues linked together by α(1→4) bonds
• Usual conformation: helix with six residues per turn
• Dietary sources: Banana, root vegetables, grains
 2. Amylopectin (80%-87%)
• Branched chains consist of 24 to 30 glucose residues with
α (1→4) linkages in the chains and by α (1→6) linkages at
the branch points
• Dietary sources: glutinous rice, waxy potato starch, waxy
corn
 Glycogen
• Storage polysaccharides in animals, hence called animal
starch
• Highly branched structure (more than amylopectin)
• Stored in muscle (β-particles, spherical) and liver
(aggregated β-particles, rosettes)
• 12-14 α-Dglucopyranose residues chains with α (1-4)
linkage (in the chain) and α (1- 6) linkages (branch)
• More branched than starch
• Less osmotic pressure
• Easily mobilized
Glycoproteins
Glycoproteins contain carbohydrate residues in addition to the
polypeptide chain.
• Immune response; for example, antibodies, which bind
to and immobilize antigens (the substances attacking
the organism), are glycoproteins.
• Carbohydrates also play an important role as antigenic
determinants, the portions of an antigenic molecule
that antibodies recognize and to which they bind.
• As antigenic determinants found in human blood
groups.
• Glycoproteins also play an important role in eukaryotic
cell membranes. The sugar portions are added to the
protein as it passes through the Golgi on its way to the
cell surface. Those glycoproteins with an extremely high
carbohydrate content (85%–95% by weight) are
classified as proteoglycans.
Hurler’s syndrome in which the material that accumulates
includes large amounts of amino sugars
Carbohydrate Digestion
• Primary sites of dietary CHO digestion are the mouth and
intestinal lumen
• Digestion is catalyzed by glycoside hydrolases (glycosidases)
that hydrolyze the glycosidic bonds
• Final products of CHO digestion are monosaccharides
(because the body can only absorb CHOs in its simplest
form)
 Glucose, galactose, and fructose which are absorbed
by cells of the small intestine
Salivary and Pancreatic α-Amylase
• Amylase is found in saliva and breaks starch into maltose
and dextrin
 This form of amylase is called ptyalin
4
• Breaks large, insoluble starch molecules into soluble
starches (amylodextrin, erythrodextrin, and achrodextrin)
• In the mouth
 Mastication mixes food with saliva, which contains
salivary α-amylase
 α-Amylase is an endoglucosidase, internal α-1, 4-bonds
between glycosyl residues in polysaccharide chains at
random intervals
• In the stomach: no amylase activity because of high acidity,
CHO digestion temporarily stops
• In the small intenstine (duodenum): pancreatic α-amylase
 Secreted by the pancreas
 Continues to hydrolyze starches and glycogen into
maltose (disaccharide), maltotriose, and oligosaccharides
 Also attacks internal α-1,4 -bonds
Absorption of Sugars
• The bulk of the dietary sugars (monosaccharides) is
absorbed in the duodenum and jejunum
• Galactose and glucose are absorbed via an active,
energyrequiring process that involves the uptake of sodium
ions
 The transport protein is the sodium-dependent glucose
cotransporter 1 (SGLT-1)
• Fructose uptake requires sodium-Independent
monosaccharide transporter (GLUT 5)
• All three of these monosaccharides are transported from
the intestinal mucosal cell into the portal circulation
(towards the liver) via GLUT 2
• Absorption by the intestinal epithelium
 Glucose is transported to the absorptive cells of the
intestine via facilitated diffusion by sodium-dependent
facilitated transport
Carbohydrates Diseases
1. Lactose Intolerance
• Decreased ability to digest lactose
• Due to lack of enzyme lactase (β-galactosidase)
• Lactose is not hydrolyzed so it is acted uponm by intestinal
bacteria causing gas flatulence
• Primary: when the amount of lactase declines as people age
• Secondary: lactose intolerance is due to injury to the small
intestine (may be from infection, kwashiorkor, colitis,
inflammatory bowel disease, etc.)
2. Fructose Intolerance
• Inability to absorb fructose efficiently
• Cause: abnorma lities in fructose transporter (GLUT 5), not
due to an enzyme
Hereditary Fructose Intolerance
 Autosomal recessive disorder
 Cause: fructose aldolase B deficiency due to lack in
any of the following enzymes
 Aldolase A – peripheral tissue
 Fructokinase – peripheral tissue
  Aldolase B – liver
 Leads to colonic bacteria metabolism of fructose and
the generation of organic acids and gases
3. Sucrose Intolerance
• Rare genetic deficiency of sucrose
• Also known as sucrase-isomaltase deficiency
• Attributed to isomaltose and starch intolerance, which
leads to diarrhea and flatulence
4. Galactosemia
• Failure of metabolizing galactose
• Leads to cataracts
• Classic Galactosemia (Type I): most common and most
severe form
• Type II Galactosemia (Galactokinase Deficiency):
accumulation of galactose or galactitol
• Type III Galactosemia (Galactose Epimerase Deficiency)
5. Hypoglycemia and Hyperglycemia
• Hypoglycemia: low blood glucose levels
• Hyperglycemia: high blood glucose levels
6. Mannosidosis
• Rare and hereditary lysosomal storage disorder
• α-Mannosidosis: defective α-monnosidase (for breakdown
of complex sugars derived from glycoproteins in the
lysosome)
• β-Mannosidosis: decreased activity of β-monnosidase (for
oligosaccharide metabolism)
7. Glucose-Galactose Malabsorption (GGM)
• Mutation of the gene that encodes Na+ -glucose co
transporter
• SLC5A1 gene: for production of SGLT1
Summary
Carbohydrates are most abundant biological molecule on earth.
Produced from CO2 fixation during Photosynthesis.
What is unique about the structures of sugars?
The simplest examples of carbohydrates are monosaccharides,
compounds that each contain a single carbonyl group and two
or more hydroxyl groups. Monosaccharides frequently
encountered in biochemistry are sugars that contain from three
to seven carbon atoms. Sugars contain one or more chiral
centers; the configurations of the possible stereoisomers can
be represented by Fischer projection formulas.
What happens if a sugar forms a cyclic molecule? Sugars exist
predominantly as cyclic molecules rather than in an open-chain
form. Haworth projection formulas are more realistic
representations of the cyclic forms of sugars than are Fischer
projection formulas. Many stereoisomers are possible for five-
and six-carbon sugars, but only a few of the possibilities are
encountered frequently in nature.
What are some oxidation–reduction reactions of sugars?
Monosaccharides can undergo various reactions. Oxidation
reactions make up one important group.
What are some important esterification reactions of sugars?
Esterification of sugars to phosphoric acid plays an important
role in metabolism.
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What are glycosides, and how do they form? The most
important reaction of sugars by far is the formation of glycosidic
linkages, which give rise to oligosaccharides and
polysaccharides.
What are some other important derivatives of sugars? Amino
sugars are the basis of cell wall structures.
What makes sucrose an important compound? Three important
examples of oligosaccharides are the disaccharides sucrose,
lactose, and maltose. Sucrose is common table sugar. It is a
disaccharide formed when a glycosidic bond forms between
glucose and fructose.
Are any other disaccharides important to us? Lactose occurs in
milk, and maltose is obtained via the hydrolysis of starch.
How do cellulose and starch differ from one another? In
polysaccharides, the repeating unit of the polymer is frequently
limited to one or two kinds of monomer. Cellulose and starch
differ in the anomeric form of
their glycosidic bonds: the α form in starch and the β form in
cellulose.
Is there more than one form of starch? Starch exists in two
polymeric forms, the linear amylose and the branched
amylopectin.
How is glycogen related to starch? Starch, found in plants, and
glycogen, which occurs in animals, differ from each other in the
degree of branching in the polymer structure.
What is chitin? Cellulose and chitin are polymers based on
single kinds of monomer units—glucose and N-
acetylglucosamine, respectively. Both polymers play structural
roles in organisms.
What role do polysaccharides play in the structure of cell walls?
In bacterial cell walls, polysaccharides are cross-linked to
peptides. Plant cell walls consist primarily of glucose.
Do polysaccharides play any specific roles in connective tissue?
Glycosaminoglycans are a type of polysaccharide based on a
repeating disaccharide in which one of the sugars is an amino
sugar and at least one of them has a negative charge owing to
the presence of a sulfate group or a carboxyl group. They play
a role in joint lubrication and in the blood clotting process.
How are carbohydrates important in the immune response? In
glycoproteins, carbohydrate residues are covalently linked to
the polypeptide chain. Such glycoproteins can play a role in the
recognition sites of antigens. A common example is the ABO
blood group, in which the three major blood types are
distinguished by sugar molecules attached to the protein
END.
 Adamson University
SY 2018 – 2019 Carbohydrates 2 May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Martinez, L., Burca, M.

Outline of the Lecture Glucose-6-Phosphate – central metabolite in the synthesis and

I. Glycogen definition decomposition of glycogen.
II. Formation of Glycogen
III. Metabolism of Glycogen
IV. Gluconeogenesis Step 3. Formation of UDP-Glucose
V. Pentose Phosphate Pathway
VI. Case Study
VII. Questions

Glycogen Definition
After a meal with high carbohydrates, what happens to the
supply of glucose that exceeds our immediate need? Glycogen formation requires energy.
➢ The body store glucose as a polymer,
• Nucleoside Triphosphate (UTP) – provides energy by
Glycogen.
hydrolysis
Linkage: α-1,4-Glycosidic bonds • UDP-glucose pyrophosphorylase – enzyme used for
Branching: α-1,6-Glycosidic bonds reaction of glucose-1-phosphate with UTP to produce
uridine diphosphate glucose (also called UDP-glucose
or UDPG) and pyrophosphate (PPi)

Step 4. Addition of UDPG to a growing chain of Glycogen.

Formation of Glycogen
Step 1. Shared step with glycolysis, includes the trapping of
glucose inside cell and committing to cellular metabolism.
– Synthesis of glycogen requires the formation of α-1,6-
Glycosidic bonds as well as α-1,4-Glycosidic bonds
glycosidic linkages using a branching enzyme.
– Each step involves formation of a new α-1,4-glycosidic
bond in a reaction catalyzed by the enzyme glycogen
synthase.

Breakdown of Glycogen
Step 2. Shared reaction with galactose metabolism
At low levels of glucose in blood:

produces Glycogen

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 Three reaction play roles in the conversion of glycogen to Phosphorylase a - phosphorylated form of glycogen
glucose-6-phosphate shown below: phosphorylase.

Step 1. Glucose residue cleaved from glycogen reacts with Phosphorylase b - dephosphorylated form of glycogen
phosphate to give glucose-1-phosphate by phosphorolysis. phosphorylase.
The reaction is catalyzed by the enzyme glycogen
phosphorylase.
In LIVER:
Glucose is an allosteric
inhibitor of phosphorylase a.
Glucose binds to the
substrate site of
phosphorylase a.
Favors the transition to
the T state.
Glucose exposes the
phosphorylated serines so
Step 2. Glucose-1-phosphate isomerizes to give glucose-6-
that the phosphatase can
phosphate. Catalyzed by the enzyme phosphoglucomutase.
hydrolyze them.
Shifts the equilibrium to
phosphorylase b.
In MUSCLES:
Primary allosteric
effectors are ATP, AMP, and
glucose-6-phosphate (G6P).
AMP stimulates
formation of the R state of
phosphorylase b, which is
active.
ATP or glucose-6-
phosphate shifts the
equilibrium back to the T
form.
[AMP], [G6P], and
[ATP] leads glycogen
degradation.
[AMP], [G6P], and
[ATP], no glycogen
degradation.

Insulin and Glucagon – hormones that regulate the key

enzyme in glycogenesis, Glycogen synthase by allosteric
Step 3. The breakdown of glycogen using a debranching effectors.
enzyme. The enzyme transfers three α-1,4-linked glucose
residues from a limit branch to the end of an other branch. Insulin Protein Phosphatase-1
activates
The same enzyme also catalyzes the hydrolysis of the α-1,6-
linked residue at the branch point.
Protein phosphatase-1 - dephosphorylates glycogen synthase
and activates it, which results in the stimulation of
glycogenesis

Gluconeogenesis
Enzymatic differences between glycolysis and
gluconeogenesis:

Glycolysis Gluconeogenesis
Regulation of Glycogen Metabolism
 Hexokinase  Glucose 6-phosphatase
Glycogen Phosphorylase
 Phosphofructokinase  Fructose-1,6-
– Major controlling factor in glycogen metabolism;  Pyruvate Kinase bisphosphatase
– For allosteric control and covalent modification.  Pyruvate carboxylase
– The enzyme is a dimer that exists in two forms, the  Phosphoenolpyruvate
inactive T (taut) form and the active R (relaxed) form. carboxykinase
Both pathways share the remaining enzymes.
Phosphorylase Kinase – catalyzes the esterification of the
serines to phosphoric acid. Step 1. Reaction of pyruvate and carbon dioxide to give
oxaloacetate. This step requires energy, which is available
Phosphoprotein Phosphatase – catalyzes the from the hydrolysis of ATP. The enzyme that catalyzes this
dephosphorylation of the serines to phosphoric acid. reaction is pyruvate carboxylase, an allosteric enzyme found in

7
 2+ Other reactions that differ from glycolysis:
the mitochondria. It also requires Magnesium ion (Mg ) and
biotin 1. Hydrolysis of fructose-1,6-bisphosphate to produce
fructose-6-phosphate and phosphate ion. Catalyzed
by the enzyme fructose-1,6-bisphosphatase, an
allosteric enzyme strongly inhibited by adenosine
monophosphate (AMP) but stimulated by ATP.

Step 2. Conversion of oxaloacetate to phosphoenolpyruvate.

The reaction is catalyzed by the enzyme phosphoenolpyruvate
carboxykinase (PEPCK), which is found in the mitochondria and
the cytosol. This reaction also involves hydrolysis of a
nucleoside triphosphate—GTP, in this case, rather than ATP.

2. The hydrolysis of glucose-6-phosphate to glucose and

phosphate ion. The enzyme that catalyzes this
reaction is glucose-6-phosphatase.

Summary of Gluconeogenesis:

Penthose Phosphate Pathway

– A pathway in sugar metabolism that gives rise to five-
carbon sugars and NADPH.
– An alternative route for the metabolism of glucose.
– Also known as Hexose Monophosphate shunt
– Occurs in cytosol
– Carried out in two steps (irreversible oxidative phase
and reversible nonoxidative phase).

Oxidative Phase

Step 1. Dehydrogenation of glucose-6phosphate to 6-

phosphogluconate catalyzed by glucose-6-phosphate
dehydrogenase.

Step 2. Hydrolysis of 6-phosphogluconolactone to Ribulose-5-

phosphate catalyzed by 6-phosphogluconate dehydrogenase.

Step 3. Formation of the ketopentose ribulose-5-phosphate.

8
Nonoxidative Phase Summary of Pentose Phosphate Pathway

Ribulose-5-phosphate - substrate for two enzymes:

1. Ribose-5-phosphate ketoisomerase - ribulose 5-

phosphate to the corresponding ribose-5- phosphate
- used for nucleotide and nucleic acid synthesis
2. Ribulose-5-phosphate 3-epimerase - alters the
configuration about carbon giving xylulose 5-
phosphate

Step 1.

Xylulose-5-phosphate (5c) and

Ribose-5- phosphate (5c)

Transketolase

Glyceraldehyde- 3-phosphate (3c) and

Sedoheptulose-7- phosphate (7c)
Step 2.
Glyceraldehyde-3-phosphate (3c) and
Sedoheptulose-7-phosphate (7c)
Transaldolase
Case Study
The pentose phosphate pathway is the only source of NADPH
in red blood cells, which, as a result, are highly dependent on
the proper functioning of the enzymes involved. A glucose-6-
phosphate dehydrogenase deficiency leads to an NADPH
deficiency, which can, in turn, lead to hemolytic anemia
because of wholesale destruction of red blood cells.

The relationship between NADPH deficiency and anemia is an

Fructose-6-phosphate and Erythrose-4- indirect one. NADPH is required to reduce the peptide
phosphate glutathione from the disulfide to the free thiol form.
Mammalian red blood cells lack mitochondria, which host many
Step 3. redox reactions.
Erythrose-4-phosphate and Xylulose-5-
Consequently, these cells are limited in the ways in which they
phosphate
can deal with redox balance. A substance like glutathione,
Transketolase which can take part in redox reactions, assumes greater
importance than would be the case in cells with large numbers
Fructose-6-phosphate and Glyceraldehyde- of mitochondria. The presence of the reduced form of
3- phosphate glutathione is necessary for the maintenance of the sulfhydryl
groups of hemoglobin and other proteins in their reduced
forms, as well as for keeping the Fe(II) of hemoglobin in its
Coenzyme for the reaction: Mg2+ and thiamin diphosphate reduced form.
(vitamin B1)
Glutathione also maintains the integrity of red cells by reacting
Step 4. with peroxides that would otherwise degrade fatty-acid side
Fructose-6 –phosphate chains in the cell membrane. About 11% of African Americans
are affected by glucose-6-phosphate dehydrogenase
deficiency.
Phosphohexose isomerase
This condition, like the sickle-cell trait, leads to increased
resistance to malaria, accounting for some of its persistence in
Glyceraldehyde-3-phosphate
the gene pool in spite of its otherwise deleterious
consequences.

9
 glucose. In the oxidative reactions of the pathway,
NADPH is also produced.

5. What are the nonoxidative reactions of the pentose

phosphatepathway and why are they important?
– The nonoxidative reactions of the pentose phosphate
pathway produce five-carbon sugars, particularly
ribose. They are important when an organism has less
need for NADPH but needs the sugars.

Questions
1. How does the breakdown of glycogen take place?
– Glycogen can readily be broken down to glucose in
response to energy needs. Glycogen phosphorylase
uses phosphate to break an α-1,4- linkage, yielding
glucose-1-phosphate and a glycogen molecule shorter
by one glucose. Debranching enzyme aids in the
degradation of the molecule around the α-1,6-
linkages.

2. How is glycogen formed from glucose?

– When an organism has an available supply of extra
glucose, more than is immediately needed as a source
of energy extracted in glycolysis, it forms glycogen, a
polymer of glucose. Glycogen synthase catalyzes the
reaction between a glycogen molecule and UDP-
glucose to add a glucose molecule to the glycogen via
an α-1,4- linkage. Branching enzyme moves sections of
a chain of glucoses so that there are α-1,6 branch
points.

3. Why is oxaloacetate an intermediate in

gluconeogenesis?
– The conversion of pyruvate (the product of glycolysis)
to glucose takes place by a process called
gluconeogenesis, which is not the exact reversal of
glycolysis. Glycolysis involves three ir- reversible steps.
One of these irreversible steps is the conversion of
phosphoenolpyruvate to pyruvate. It is favorable to
convert pyruvate to oxaloacetate to facilitate the
conversion to phosphoenolpyruvate.

4. What are the oxidative reactions of the pentose

phosphate pathway?
– The pentose phosphate pathway is an alternative
pathway for glucose metabolism. In this pathway five-
carbon sugars, including ribose, are produced from
10
 Adamson University
SY 2018 – 2019 Glycolysis May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Burca, Mary T., Martinez Audrey B.

Outline of the Lecture

I. Over all Pathway of glycolysis Step 1: Phosphorylation
II. Anaerobic Metabolism of Pyruvate
III. Feeder pathway
IV. Case Study
V. Questions

Over all Pathway of glycolysis

Glucose is a key metabolite in human metabolism. The first step
in the degradation of glucose is glycolysis, which breaks down
glucose to pyruvate. The main purpose of glycolysis- (from the
Greek glykys, meaning “sweet,” and lysis, meaning “splitting”)
is the generation of energy (ATP). A modest amount of ATP is
produced in glycolysis directly, but much more ATP is formed
downstream of glycolysis through the complete oxidation of
pyruvate.
Glucose is phosphorylated by ATP to form a sugar phosphate.
The negative charge of the phosphate prevents passage of the
sugar phosphate through the plasma membrane, trapping
glucose inside the cell.
Step 2: Isomerization

A readily reversible rearrangement of the chemical structure

(isomerization) moves the carbonyl oxygen from carbon 1 to
carbon 2, forming a ketose from an aldose sugar.
Figure 1-1 Major pathways of glucose utilization. Although not
the only possible fates for glucose, these three pathways are Step 3: Phosphorylation
the most significant in terms of the amount of glucose that
flows through them in most cells.

An Overview: Glycolysis Has Two Phases

Phase 1
The new hydroxyl group on carbon 1 is phosphorylated by ATP,
(Energy Investment)
in preparation for the formation of two three-carbon sugar
Glucose molecule is
phosphates. The entry of sugars into glycolysis is controlled at
split, using 2 ATP
this step, through regulation of the enzyme
phosphofructokinase.

Step 4: Cleavage

Phase 2
(Energy Payoff)
Energy is extracted
in the form of 4 ATP

Details of the 10 steps of glycolysis

11
The six-carbon sugar is cleaved to produce two three-carbon Step 9: Dehydration
molecules. Only the glyceraldehyde 3phosphate can proceed
immediately through glycolysis.

Step 5: Isomerization

The removal of water from 2-phosphoglycerate creates a high-

energy enol phosphate linkage.

Step 10: Substrate level Phosphorylation

The other product of step 4, dihydroxyacetone phosphate, is

isomerized to form glyceraldehyde 3-phosphate.

Step 6: Oxidation

The transfer to ADP of the high-energy phosphate group that

was generated in step 9 forms ATP, completing glycolysis.

ATP Formation Coupled to

Glycolysis During glycolysis
The two molecules of glyceraldehyde 3-phosphate are oxidized. some of the energy of the
The energy-generation phase of glycolysis begins, as NADH and glucose molecule is Glucose + 2NAD + 2ADP + 2Pi
a new high-energy anhydride linkage to phosphate are formed conserved in ATP, while >> 2 pyruvate + 2NADH +
much remains in the 2H2O + 2ATP +2H2O
Step 7: Substrate level phosphorylation product, pyruvate. The
overall equation for
glycolysis is

For each molecule of glucose

degraded to pyruvate, two
molecules of ATP are
generated from ADP and Pi. Glucose + 2NAD >> 2
The transfer to ADP of the high-energy phosphate group that We can now resolve the pyruvate + 2NADH + 2H+
was generated in step 6 forms ATP. equation of glycolysis into
two processes the ΔG°= -146 kJ/mol
Step 8: Shift of phosphoryl group conversion of glucose to
pyruvate, which is exergonic:

The remaining phosphate ester linkage in 3-phosphoglycerate,

which has a relatively low free energy of hydrolysis, is moved
from carbon 3 to carbon 2 to form 2-phosphoglycerate.

12
 Three possible catabolic fates of the pyruvate formed in
Three actions of glycolysis are the regulatory steps
glycolysis. Pyruvate also serves as a precursor in many anabolic
1. Conversion of glucose to glucose-6-phospahte reactions.
catalyzed by hexokinase (1)
The three immediate metabolic fates of pyruvate are:
2. Fructose to fructose 1, 6- bisphosphate catalyzed by
fructokinase (3) 1. Converted to ethanol (anaerobic)
3. Formation of pyruvate from phospho-enol-pyruvate
(PEP) catalyzed by kinase (10) 2. Converted to lactate (anaerobic)

3. Converted to Acetyl CoA

Anaerobic Metabolism of Pyruvate
Fates of Pyruvate With the exception of some interesting
variations in the bacterial realm, the pyruvate formed by
glycolysis is further metabolized via one of three catabolic
routes. In aerobic organisms or tissues, under aerobic
conditions, glycolysis is only the first stage in the complete
degradation of glucose.
Anaerobic fates for pyruvate is important because NAD+ needs
to be regenerated for glycolysis to take place in anaerobic
conditions. Lactate form in muscle cells, and ethanol in yeast
cells through a process called fermentation.
The production of lactate:

• Lactate is not an acid

• It is a dead-end product in skeletal muscles during

anaerobic activity

• A membrane transporter protein transports the

lactate out of the muscle and into the blood.

• The pH of the blood is lowered, O2 is released from

hemoglobin

• Muscle cell gets more oxygen

• Under aerobic conditions, lactate is a metabolic fuel

for cardiac issue.

• Hydrolysis of ATP by myosin produces hydrogen

which can cause acidoic damage to muscle ibres, not
lactate.
Figure 1-1 Fates of Pyruvate

13
 The production of ethanol:
Pyruvate Alcohol
decarboxylase dehydrogenase
• Does not occur in vertebrates
• Occurs in yeast
• Pyruvate is decarboxylated and reduced to form
ethanol
• In the process one molecule of CO2, NAD+ and
ethanol is produced
Production of Acetyl-CoA:
• Acetyl CoA links glycolysis to the citric acid cycle.
• The conversion of pyruvate to acetyl CoA happens in
the mitochondrial matrix
• the reaction is catalyzed by pyruvate dehydrogenase
complex
• converted via the pyruvate dehydrogenase complex
• One molecule of CO2 and NADH is also generated in
the process
• Acetyl- CoA is a Coenzyme A bonded to acetyl group
with a thioester bond
• CoA is a derivative of vitamin B5 linked to an
adenosine nucleotide. Its functional group is SH (thiol)
• The formation of acetyl-CoA is irreversible in
carbohydrate metabolism
• It is reversible in lipid metabolism
• the pyruvate dehydrogenase reaction requires 5
cofactors including NAD+, FAD and CoA
• It is an example of oxidative decarboxylation
• Pyruvate hydrogenase is a multi-enzyme complex (3
catalytic enzymes) (speeds up reaction time, limits
14
number of side reaction, enzyme controlled as single
unit)
• Acetyl CoA is a high energy intermediate
Transportation of pyruvate from cytosol to mitochondrial
matrix: Pyruvate passes through porins in the outer membrane
of the mitochondria Pyruvate passes through the transport
protein pyruvate translocase in the inner membrane of the
mitochondria Pyruvate translocase is a symport. A proton also
enter matrix with the pyruvate molecule.
Regulation of pyruvate dehydrogenase:
1. NAD+/NADH: NADH inhibits PDH by protein kinase
activation (phosphorylation of PDH, allosteric)
2. Acetyl Co-A: Inhibitor, activates protein kinase
(phosphorylation of PDH)
3. Ca2+: Activator, dephosphorylates PDH protein
phosphatase activation)
PDH is regulated by reversible phosphorylation. It is switched
of when energy level is very high (Ratio of ATP/ADP is high).
Phosphorylation of PDH witches of its activity and vice versa.
Protein kinase is responsible for phosphorylation of PDH.
Protein kinase is activated by NADH and acetyl CoA. It is
inhibited by NAD+, pyruvate and ADP.
FEEDER PATHWAY
Many carbohydrates besides glucose meet their catabolic fate
in glycolysis, after being transformed into one of the glycolytic
intermediates. The most significant are the storage
polysaccharides glycogen and starch; the disaccharides
maltose, lactose, trehalose, and sucrose; and the
monosaccharides fructose, mannose, and galactose
• Glycogen and starch, polymeric storage forms of
glucose, enter glycolysis in a two-step process.
Phosphorolytic cleavage of a glucose residue from an
end of the polymer, forming glucose 1-phosphate, is
catalyzed by glycogen phosphorylase or starch
phosphorylase. Phosphoglucomutase then converts
the glucose 1-phosphate to glucose 6-phosphate,
which can enter glycolysis.
• Ingested polysaccharides and disaccharides are
converted to monosaccharides by intestinal
hydrolytic enzymes, and the monosaccharides then
enter intestinal cells and are transported to the liver
or other tissues.
• A variety of D-hexoses, including fructose, galactose,
and mannose, can be funneled into glycolysis. Each is
phosphorylated and converted to either glucose 6-
phosphate or fructose 6-phosphate.
• Conversion of galactose 1-phosphate to glucose 1-
phosphate involves two nucleotide derivatives: UDP-
galactose and UDP-glucose. Genetic defects in any of
the three enzymes that catalyze conversion of
galactose to glucose 1-phosphate result in
galactosemias of varying severity.

Figure 1- 3 Feeder pathway

Figure 1-2 hydrolysis of Disaccharides

15
 CASE STUDY

Galactose and fructose disorders

Disorders Enzyme Effects It can

lacking get
from?
Galactosemia ga UDP jaundice, milk
lactose-1- galactose lethargy
phosphate accu kidney disease,
mulates. and weight loss
Hereditary enzyme fru vomiting, hypog fruits,
fructose ctose-1- lycemia, liver table
intolerance phosphate dysfunction, sugar
(HFI) - Fructose aldolase and kidney defe (sucro
1,6- cts se),
diphosphatase and
deficiency is infant
associated with formul
an impaired as
ability to form contai
glucose from ning
other substrates sucros
(a process e.
called gluconeo
genesis)

SAMPLE QUESTIONS

1. Name the 9th enzyme use in glycolysis

2. Net ATP produce?
3. Regulatory Steps
4. What is gluconeogenesis?
5. Name the 3 fates of pyruvates

Answer
1. Enolase
2. 2 ATP
3. 1,3 and 10
4. The glycolytic pathway can be used for the synthesis
of glucose from simpler molecules
5. Formation of Acety-CoA, Fermentation and Lactate.

16
 Adamson University
SY 2018 – 2019 Citric Acid Cycle May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc Yoshimi N. Manuel

Outline of the Lecture

I. Citric Acid Cycle
II. Cellular Respiration
III. Production of Acetyl CoA
IV. Step-by-step Reactions of Citric Acid Cycle
V. Regulation of the Citric Acid Cycle

Citric Acid Cycle

– Also called the Tricarboxylic Acid Cycle (TCA) and Krebs
cycle, in honor of Hans Adolf Krebs.
– In eukaryotes, the citric acid cycle takes place in the
matrix of the mitochondria, just like the conversion of
pyruvate to acetyl CoA. In prokaryotes, these steps both
take place in the cytoplasm. The citric acid cycle is a
closed loop; the last part of the pathway reforms the
molecule used in the first step.
– The citric acid cycle is a hub in metabolism, with
degradative pathways leading in and anabolic pathways
leading out, and it is closely regulated in coordination
with other pathways. The cycle includes eight major Figure 2. Catabolism of proteins, fats, and carbohydrates
in the three stages of cellular respiration.
steps.
Stage 1: Acetyl CoA production – Organic fuels (Glucose,
amino acids, and fats) yield Acetyl CoA.
Stage 2: Acetyl CoA enters citric acid cycle and is enzymatically
oxidized; energy is conserved in electron carriers, NADH and
FADH2.
Stage 3: Energy reach in electron from NADH and FADH2
reduce O2 to H2O.

Production of Acetyl CoA

Figure 1. Citric Acid Cycle

Cellular Respiration
– It is the process by which organisms combine oxygen with
foodstuff molecules, diverting the chemical energy in
these substances into life-sustaining activities and
discarding, as waste products, carbon dioxide and water.
– In the broader physiological or macroscopic sense, Figure 3. Formation of Acetyl CoA from Pyruvate catalyzed by
respiration refers to a multicellular organism’s uptake of Pyruvate Dehydrogenase Complex.
O2 and release of CO2. Biochemists and cell biologists,
however, use the term in a narrower sense to refer to the – Before entering the citric acid cycle, the carbon skeletons
molecular processes by which cells consume O2 and of sugars and fatty acids are degraded to the acetyl group
produce CO2—processes more precisely termed cellular of Acetyl CoA, the form in which the cycle accepts most of
respiration. its fuel input. Acetyl CoA is an acetate attached to
– Cellular respiration has three phases: Acetyl CoA Coenzyme A.
production, Acetyl CoA oxidation, and Electron transfer.

17

Figure 4. Coenzyme A.
– The Pyruvate Dehydrogenase Complex links glycolysis to
the Citric acid cycle. It is a large multi-enzyme complex
composed of three enzymes involving five cofactors. The
pyruvate dehydrogenase complex oxidizes pyruvate to
generate Acetyl CoA.
– The Pyruvate Dehydrogenase Complex requires five
coenzymes:
• Thiamine pyrophosphate (TPP)
• Flavin adenine dinucleotide (FAD)
• Coenzyme A (CoA, sometimes denoted CoA-SH,
to emphasize the role of the -SH group)
• Nicotinamide adenine nucleotide (NAD)
• Lipoate
– It also Consists of three distinct enzymes:
• Pyruvate dehydrogenase (E1)
• Dihydrolipoyl transacetylase (E2)
• Dihydrolipoyl dehydrogenase (E3)
Figure 6. Formation of Isocitrate from Citrate catalyzed by cis-
Figure 5. Oxidative decarboxylation of pyruvate to Acetyl-
CoA by the PDH complex.
Step-by-step Reactions of Citric Acid Cycle
1. Formation of Citrate - Oxaloacetate + Acetyl-CoA
+ H2O Citrate + CoA
Mechanism: Claisen condensation reaction from organic
chemistry, where thioester is combined with ketone.
Methyl carbon of acetylCoA attack the electron poor
ketone carbon of oxaloacetate. You have to abstract a
18
proton, so you produce a carbanion here. The carbanion
will attack this oxaloacetate carbonyl carbon. To produce
carbanion, the enzyme uses the general acid/base
mechanism with two His residues.
Figure 5. Formation of Citrate from Acetyl CoA and
Oxaloacetate catalyzed by Citrate Synthase.
2. Formation of Isocitrate via cis-Aconitate – Citrate
[H2O + cis-Aconitate ] Isocitrate
Mechanim: This is a dehydration reaction followed by a
hydration. The dehydration step is like enolase in
glycolysis. To facilitate the abstraction of the proton, you
pull the electrons strongly from the carboxylate. Enolase
did this by putting magnesium near the carboxylate
oxygen. Aconitase uses an iron-sulfur cluster cofactor
instead of magnesium to do this. Three Cys residues and
multiple Fe atoms make the cluster.
Aconitate.
3. Oxidation of Isocitrate to α-Ketoglutarate and CO2 -
Isocitrate α-keto glutarate + CO2
Mechanism: After hydride transfer, the enzyme uses a
Mn2+ -ion cofactor. The metal further enhances the
electron withdrawing power of the carbonyl, facilitating
decarboxylation. α-keto glutarate is a β-keto carboxylic
acid. β-keto carboxylic acids are known to be unstable
and have a natural tendency to release this carboxylic
acid group as CO2.
 6. Oxidation of Succinate to Fumarate

Mechanism: Electrons pass from succinate through the

FAD and iron-sulfur centers before entering the chain of
electron carriers in the mitochondrial inner membrane (or
the plasma membrane in bacteria). Electron flow from
succinate through these carriers to the final electron
Figure 7. Formation of α-Ketoglutarate from Isocitrate acceptor, O2, is coupled to the synthesis of about 1.5 ATP
catalyzed by Isocitrate Dehydrogenase. molecules per pair of electrons (respiration-linked
phosphorylation).
4. Oxidation of α-ketoglutarate to Succinyl CoA and CO2 -
α-keto glutarate + CoA Succinyl-CoA

Mechanism: α-keto glutarate dehydrogenase works

exactly like pyruvate dehydrogenase. You have the five
coenzymes: TPP, lipoyllysine, CoA, FAD and NAD+. These
are all used, and you get oxidation. The decarboxylated
product occurs as a thioester. The product is succinyl-
CoA. The thioester in the succinyl-CoA will be utilized later
of course in an analogous manner.

Figure 10. Formation of Fumarate from Succinate catalyzed by

Succinate Dehydrogenase.

7. Hydration of Fumarate to Malate

Mechanism: This enzyme is highly stereospecific; it

catalyzes hydration of the trans double bond of fumarate
but not the cis double bond of maleate (the cis isomer of
fumarate). In the reverse direction (from L-malate to
fumarate), fumarase is equally stereospecific: D-malate is
not a substrate.
Figure 8. Formation of Succinyl CoA from α-Ketoglutarate
catalyzed by α-Ketoglutarate Dehydrogenase Complex.

5. Conversion of Succinyl CoA to Succinate - Succinyl-

CoA + GDP + Pi Succinate + GTP

Mechanism: Phosphorylysis reaction is followed by

phosphoryl transfer to GDP, producing succinate plus
GTP. Note the phosphoryl group is transferred to the GDP
via an intermediate that forms with a His residue on the
enzyme’s active site.

Figure 11. Formation of Malate from Fumarate catalyzed

by Fumarase.

Figure 9. Formation of Succinate from Succinyl CoA

catalyzed by Succinyl CoA Synthetase.
19
 8. Oxidation of Malate to Oxaloacetate
Mechanism: The equilibrium of this reaction lies far to the
left under standard thermodynamic conditions, but in
intact cells This keeps the concentration of oxaloacetate
in the cell extremely low ( 106 M), pulling the malate
dehydrogenase reaction toward the formation of
oxaloacetate. oxaloacetate is continually removed by the
highly exergonic citrate synthase reaction.
Figure 12. Formation of Oxaloacetate from L-Malate
catalyzed by Malate Dehydrogenase.
Figure 13. Overall net reaction of the Citric acid cycle.
Regulation of the Citric Acid Cycle
– Production of Acetyl CoA by the Pyruvate
Dehydrogenase Complex is regulated by Allosteric
and covalent mechanism.
– The PDH complex of mammals is strongly inhibited by
ATP and by acetyl-CoA and NADH, the products of
the reaction catalyzed by the complex. The allosteric
inhibition of pyruvate oxidation is greatly enhanced
when long-chain fatty acids are available. AMP, CoA,
and NAD, all of which accumulate when too little
acetate flows into the citric acid cycle, allosterically
activate the PDH complex. Thus, this enzyme activity
is turned off when ample fuel is available in the form
of fatty acids and acetyl-CoA and when the cell’s
[ATP]/[ADP] and [NADH]/[NAD] ratios are high, and it
is turned on again when energy demands are high
and the cell requires greater flux of acetyl-CoA into
the citric acid cycle.
– It is regulated at its three exergonic state:
20
• Substrate availability
• Inhibition by accumulating products
• Allosteric feedback inhibition of the
enzymes that catalyze early steps in the
cycle.
Figure 14. Regulation of metabolite flow from the PDH
complex through the citric acid cycle.
SUMMARY:
The tricarboxylic acid cycle is the major energy-
yielding metabolic pathway in cells, providing the greater part
of the reduced coenzymes that will be oxidized by the electron
transport chain to yield adenosine triphosphate (ATP). The
pathway is sometimes known as the citric acid cycle, or the
Krebs' cycle, after its discoverer, Sir Hans Krebs. In addition to
its role in energy-yielding metabolism, and the oxidation of 2-
carbon units, the cycle is also the major pathway for
interconversion of 4- and 5-carbon compounds in the cell,
many of which arise from, or are intermediates in the
synthesis of, amino acids. Oxaloacetate, a key intermediate in
the cycle, is the main precursor for gluconeogenesis in
the fasting state.
The pyruvate dehydrogenase complex catalyzes an irreversible
reaction that is the entry point of pyruvate into the TCA cycle
and is under complex regulation by allosteric and covalent
modification of the pyruvate dehydrogenase component of
the complex. The end products of the overall reaction (NADH
and acetyl-CoA) are potent allosteric inhibitors of the pyruvate
dehydrogenase component of the complex.
 Adamson University
SY 2018 – 2019 Electron Transport Chain May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. John Eric Pamaran

Outline of the Lecture • Then transfer to a lipid-soluble carrier,

I. Electron Transport Chain Ubiquinone (Q).
II. Complex I: NADH Dehydrogenase
• Pump 4 H+ ions to Inter Mitochondrial Matrix.
III. Complex II: Succinate Dehydrogenase
IV. Complex III: Cytochrome Reductase
V. Complex IV: Cytochrome C oxidase
VI. Last Step: ATP Synthase (Complex V)
VII. Clinical Significance of Electron Transport Chain

Electron Transport Chain

Electron Transport Chain (ETC) is a chemical reaction between

an electron donor (such as NADH) and an electron acceptor
(such as O2) to the transfer of H+ ions across a membrane,
through a set of mediating biochemical reactions.

Complex II: Succinate Dehydrogenase

• Do not pump H+ ions to Inter Mitochondrial
Matrix.
• Catalyzes the conversion of succinate to
fumarate. FAD oxidizes succinate to fumarate
(FAD becoming reduced to FADH2 as it picks up
two electrons and two protons).
• The electrons are then passed to Co-Q. It
diffuses easily and shuttles the electrons to
complex III.

Location: Mitochondrial Membrane

Contains Four Enzyme Complex:

Complex I: NADH Dehydrogenase
Complex II: Succinate Dehydrogenase
Complex III: Cytochrome Reductase
Complex IV: Cytochrome C oxidase

Last Step: ATP synthase produce ATP using the proton gradient
in Inter Mitochondrial Membrane.
Complex III: Cytochrome Reductase
For every, 1 NADH = 10 H+ = 2.5 ATP
Complex I: NADH Dehydrogenase
• Removes two electrons from NADH.
• The electrons are then passed to iron-sulphur
proteins (FeS) (this is non-heme iron). The
electron is accepted by Fe3+ which is reduced to
Fe2+ (Fe3+ is reduced to Fe2+ by electrons).
• Contains: Cytochrome B, Cytochrome C1 and FeS
proteins.
• Co-QH2 passes two electrons to Cytochrome B.
• Then the electrons are passed to the FeS proteins
and then to cytochrome C1.
• Cytochrome C1 pass the electrons to Cytochrome C.
• Cytochrome C transfers the electron to complex IV.
• Move 4 H+ ions to Inter Mitochondrial Matrix. (Two
protons from reduce quinone to quinol and two
protons are released from two ubiquinol molecules)

21
 Complex IV: Cytochrome C oxidase
• Contains: Cytochrome A and Cytochrome A3 (both use Fe
and Cu atoms to handle the electrons).
• Oxidizes the Cytochrome C with the help of
Cytochrome A and Cytochrome A3
• Then it would be transferred to O2 to produce water
(H20).
• Move 2 H+ ions to Inter Mitochondrial Matrix.
Last Step: ATP synthase (Complex V)
• The ETC and oxidative phosphorylation are coupled
by the ATP synthase using the proton gradient from
the inner mitochondrial membrane.
• This process is called ATP production via oxidative
phosphorylation of ADP and Inorganic Phosphorus.
• Sometimes described as Complex V of the electron
transport chain.
• Pumps back 4 H+ ions to Inner Mitochondrial
Matrix.
22
Clinical Significance of Electron Transport Chain
• Uncoupling agents: dissociation of the electron
transport chain and ATP synthase
• Increased permeability of mitochondrial membrane
→ reduced proton gradient and increased
oxygen consumption → electron transfer continues
but ATP synthesis stops
• Thermogenin (in brown fat): A proton channel that
physiologically uncouples electron transport and ATP
synthesis to generate heat.
• Poisons that disrupt oxidative phosphorylation
• Electron transport inhibitors: block ATP synthesis by
reducing the proton gradient:
- Rotenone: inhibits complex I
- Antimycin: inhibits complex III
- Cyanide, carbon monoxide, Azides: inhibits complex
IV
• ATP synthase inhibitors: block ATP synthesis by
stopping the electron transfer via an increased
proton gradient
Questions
1. Where does the electron transport chain occur?
2. What is the electron source of electron transport chain?
3. It is a lipid soluble carrier of Complex I and II?
4. What is the Net H+ ions pump during the ETC?
5. What is the final product obtained from ATP synthase?
Summary
The Electron Transport Chain is a series of redox reactions that
transfer electrons from an electron donor to an electron
acceptor (which happens in Complex I-IV). Then the H+ in the
proton gradient are coupled with the electrons. Lastly, The
ATP synthase produces the ATP needed using the ADP and the
Inorganic Phosphorus.
END.
 Adamson University May 24, 2019
SY 2018 – 2019 Lipids Ocampo, Faye Ann
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Taño, Harlan Jake

Outline of the Lecture Structural Lipids in Membranes

I. Storage Lipids Biological membranes
II. Structural Lipids in Membranes – feature a double layer of lipids, which acts as a barrier to
III. Abnormal Accumulations of Membrane Lipids
IV. Summary the passage of polar molecules and ions
V. Questions – Membrane lipids: amphipathic
– Their hydrophobic interactions with each other
Storage Lipids and their hydrophilic interactions with water
– Principal form of stored energy in most organisms and direct their packing into sheets called lipid
major constituents of cellular membranes. bilayers
– Common and defining feature is their insolubility in water
Lipid bilayer
I. Fatty acids – the polar head groups are in contact with water, and the
– carboxylic acids with hydrocarbon chains ranging from 4 nonpolar tails lie in the interior of the membrane
to 36 carbons long (C4 to C36) – nonpolar hydrocarbon interior consists of the saturated
– has a carboxyl group at the polar end and a hydrocarbon and unsaturated chains of fatty acids and the fused-ring
chain at the nonpolar tail system of cholesterol
– Naturally Occurring Fatty Acids
 Palmitic acid
 Stearic acid
 Linoleic acid
 Arachidonic acid

Polyunsaturated Fatty Acids (PUFAs)

– Has a double bond between the third and fourth carbon
from the methyl end of the chain

II. Triacylglycerols
– lipids formed by esterification of three fatty acids to
glycerol; also called a triglyceride
– form a separate phase of microscopic, oily droplets in
the aqueous cytosol, serving as depots of metabolic fuel
– storage fats found in many foods

Lipases
– enzymes that catalyze the hydrolysis of stored
triacylglycerols, releasing fatty acids for export to sites
where they are required as fuel

III. Phosphoacylglycerols
– phosphatidic acid with another alcohol esterified to the
phosphoric acid moiety

IV. Waxes
Phospholipids
– mixtures of esters of long-chain carboxylic acids and long-
– a polar head group is joined to the hydrophobic moiety
chain alcohols
by a phosphodiester linkage
– Functions:
– Planktons: chief storage form of metabolic fuel
Glycolipids
– Vertebrates: to protect hair and skin and keep it
– sphingolipids that lack phosphate but have a simple
pliable, lubricated, and waterproof
sugar or complex oligosaccharide at their polar ends

23
Five General Types of Membrane Lipids:
1. Glycerophospholipids
– also called phosphoglycerides
– two fatty acids are attached in ester linkage to the first
and second carbons of glycerol, and a highly polar or
charged group is attached through a phosphodiester
linkage to the third carbon
2. Galactolipids and sulfolipids
Galactolipids
– predominate in plant cells
– contain two fatty acids esterified to glycerol, but lack the
characteristic phosphate of phospholipids
– one or two galactose residues are connected by a
glycosidic linkage to C-3 of a 1,2-diacylglycerol
– localized in the thylakoid membranes (internal
membranes) of chloroplasts
Sulfolipids
– a sulfonated glucose residue is joined to a diacylglycerol in
glycosidic linkage
– the sulfonate group bears a negative charge like that of
the phosphate group in phospholipids
3. Archaeal tetraether lipids
– at each end of the extended molecule is a polar head
consisting of glycerol linked to either phosphate or
sugar residues.
– General name: glycerol dialkyl glycerol tetraethers
(GDGTs)
4. Sphingolipids
– have a polar head group and two nonpolar tails, but
unlike glycerophospholipids and galactolipids they
contain no glycerol
– composed of one molecule of the long-chain amino
alcohol sphingosine or one of its derivatives, one
molecule of a long-chain fatty acid, and a polar head
group that is joined by a glycosidic linkage or a
phosphodiester
24
Abnormal Accumulations of Membrane Lipids
– The breakdown of lipids is promoted by hydrolytic
enzymes in lysosomes, each enzyme capable of
hydrolyzing a specific bond
– When sphingolipid degradation is impaired by a defect
in one of these enzymes, partial breakdown products
accumulate in the tissues, causing serious disease
Niemann-Pick disease
– caused by a rare genetic defect in the enzyme
sphingomyelinase, which cleaves phosphocholine from
sphingomyelin
– Sphingomyelin accumulates in the brain, spleen, and
liver. The disease becomes evident in infants and causes
mental retardation and early death
Tay-Sachs disease
– ganglioside GM2 accumulates in the brain and spleen
owing to lack of the enzyme hexosaminidase A
– symptoms: progressive developmental retardation,
paralysis, blindness, and death by the age of 3 or 4 years
Gaucher disease
– characterized by the deposition of glucocerebroside in
cells of the macrophage-monocyte system.
– results from the deficiency of the enzyme
glucocerebrosidase
Sandhoff disease
– an autosomal recessive genetic disorder caused by an
abnormal gene for the beta subunit of the
hexosaminidase B enzyme
– results in a deficiency of hexosaminidase A and B that
results in accumulation of fats (lipids) called GM2
gangliosides in the neurons and other tissues
Fabry disease
– caused by mutations in the GLA gene, which provides
instructions for making the enzyme alpha-galactosidase
A
– mutations in the GLA gene alter the structure and
function of the enzyme, preventing it from breaking
down globotriaosylceramide
– The progressive accumulation of globotriaosylceramide
damages cells
 • True or false. Lipids are water-insoluble cellular
components, of diverse structure, that can be extracted
from tissues by nonpolar solvents. True.
• What is the second group of membrane lipids are those
that pre-dominate in plant cells in which one or two
galactose residues are connected by a glycosidic linkage
to C-3 of a 1,2-diacylglycerol? Galactolipids
• True or false. Steroid hormones are derived from sterols.
They serve as powerful biological signals, altering gene
expression in target. True.

END

Summary
 Lipids are water-insoluble cellular components, of
diverse structure, that can be extracted from tissues by
nonpolar solvents.
 Triacylglycerols are primarily storage fats; they are
present in many foods.
 The polar lipids, with polar heads and nonpolar tails, are
major components of membranes. The most abundant
are the glycerophospholipids.
 Chloroplast membranes are rich in galactolipids,
composed of a diacylglycerol with one or two linked
galactose residues, and sulfolipids, diacylglycerols with a
linked sulfonated sugar residue and thus a negatively
charged head group.
 Archaeal tetraether lipids are stable under the harsh
conditions in which archaea live.

Questions
• These are the simpliest lipids constructed from fatty
acids, also referred to as triglycerides, fats, or neutral
fats? Triacylglycerols
• The most commonly occurring fatty acids contains hoe
many carbon atoms in an unbranched chain? 12-24

25
 Adamson University
SY 2018 – 2019 LIPID CATABOLISM AND LIPID ANABOLISM May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. HERRERA & MORALES

Outline of the Lecture • Lipid-binding molecules in the blood in the blood,

I. Digestion, Mobilization & Transport of fats responsible for the transport of triacylglycerols,
II. Hormones trigger mobilization of stored phospholipids & cholesterol between organs.
triacylglycerols
III. Fatty acids are activated and transported into Hormones trigger mobilization of stored triacylglycerols
mitochondria • Before fats can be used as fuels, the triacylglycerols
IV. The four β-Oxidation steps are repeated to yield storage form must be hydrolyzed to yield isolated fatty
Acetyl-CoA & ATP acids.
V. Oxidation of Unsaturated Fatty Acids requires two • The reaction is catalyzed by a hormonally controlled
additional reactions lipase.
VI. Ketogenesis • Under physiological condition facing an early-morning
VII. Lipid anabolism runner, glucagon & epinephrine will be present.
VIII. Interaction Between Lipids
IX. Lipid Mechanism • In adipose tissue, these hormones trigger 7 TM
X. Lipid Metabolism Receptors that activate adenylate cyclase.
XI. Activation of Acetyl CoA • Increase level of cyclic AMP then stimulates protein
XII. Pentose Phosphate Pathway
XIII. Fatty Acid Synthesis kinase A, which phosphorylates two key proteins:
XIV. Lipid-Related Disease
XV. Summary (1) Perilipin
Digestion, Mobilization & Transport of fats (2) Hormone Sensitive lipase
• Triacylglycerol are highly concentrated stores of STEPS:
metabolic energy because they are reduced &
anhydrous (1) Restructures the fat droplet, so that the
• 40% or more of the daily energy requirements is triacylglycerols are more accessible to the
supplied by dietary triacylglycerols. mobilization.
• Sole energy source of hibernating animals & migrating (2) The phosphorylation of perilipin triggers the release
birds. of a coactivator for Adipose triglyceride lipase (ATGL)
(3) Once bound to the coactivator, ATGL initiates
To absorb triacylglycerol through the intestinal wall, it mobilization of triacylglycerols by releasing fatty acid
must be converted from insoluble macroscopic fat from triacylglycerol, forming diacylglycerol.
particles into soluble one. It can be solubilized by bile salts. (4) Diacylglycerol is then converted into a free fatty acid
Bile Salts (e.g taurocholic acid) & monoacylglycerol by the hormone-sensitive lipase.
(5) Finally, monoacylglycerol lipase completes the
• Synthesized from cholesterol in the liver, stored in gall mobilization of fatty acids with the production of a
bladder & released into the small intestine. free fatty acid and glycerol.
• are amphiphatic compounds that act as biological
detergents. Noted: Epinephrine & Glucagon induce lipolysis.
• Converting dietary fats into mixed micelles of bile salts
& triacylglycerols.

Chylomicrons (Transport)

• In the intestinal mucosal cells, the triacylglycerols are

resynthesized from fatty acids and
monoacylglycerols.
• Then, packaged into lipoprotein transport particle
called “chylomicrons”

Apolipoproteins

• “apo” means “detached”

26
 Fatty acids are activated and transported into mitochondria • Located on the inner face of the inner mitochondrial
• Fatty oxidation in animal cells are located in the membrane.
mitochondrial matrix. • Regenerates fatty acid acyl-CoA and releases free
• 12 or fewer carbons fatty acid can enter mitochondria carnitine into the matrix
without the help of membrane transporter.
• But 14 or more carbons cannot pass directly through
mitochondrial membranes.
• Must undergo the three enzymatic steps of the
carnitine shuttle
Acyl CoA dehydrogenase
Step Reaction Enzyme • Gives enoyl CoA with a trans double bond between C-
1 Fatty acid + CoA Acyl-CoA Acyl CoA 2 & C-3
+ PPi synthetase (fatty • FAD rather than NAD is the electron acceptor because
acid thiokinase) the ΔG for this reaction is insufficient to drive the
2 Carnitine + Acyl-CoA acyl Carnitine reduction of NAD+
carnitine + CoA acyltransferase
3 Acyl CoA + E-FAD trans- Acyl CoA
Δ2-enoyl CoA + E-FADH2 dehydrogenases
4 Trans- Δ2-Enoyl CoA + H2O Enoyl CoA
L-3-hydroxyl CoA hydratase
5 L-3-Hydroxyl CoA + NAD+ L-3-Hydroxyacyl
3-ketoacyl CoA + NADH + CoA
H+ dehydrogenase
6 3-ketoacyl-CoA + CoA β-ketothiolase
acetyl CoA + acyl CoA (thiolase)

Enoyl-CoA hydratase
Enzymes: • The 2nd step of β-Oxidation
Acyl-CoA synthetase • Converts the hydroxyl group at C-3 into a keto group
• Catalyze the formation of a thioester linkage between & generates NADH.
the fatty acid carboxyl group and the thiol group of • Specific fo the L isomer of the hydroxyacyl substrate.
Coenzyme A to yield a fatty acyl-CoA
• Then coupled to the cleavage of ATP to AMP & PPi

Carnitine Synthetase

Carnitine acyltransferase I
• Catalyzed the transesterification in the outer L-3-Hydroxyacyl CoA dehydrogenase
membrane. • L-3-hydroxyacyl CoA is dehydrogenated to form 3-
• Acyl-CoA is converted into carnitine ester on the ketoacyl-CoA, the NAD+ is the electron acceptor.
cytosolicface of the outer membrane. • The NADH formed in the reaction donates its electrons
• The fatty acyl-carnitine ester then enters the matrix by to NADH dehydrogenase, an e- carrier of the
facilitated diffusion through the acyl- respiratory chain, ATP is formed from ADP as the e-
carnitine/carnitine transporter. pass to O2.

Carnitine acyltransferase II
β-ketothiolase (thiolase)
• Fatty acyl group is enzymatically transferred from
carnitine to intramitochondrial coenzyme A • The 4th and last step of the β-oxidation cycle

27
 • Promotes reaction of β-ketoacyl-CoA with a molecule
of free Coenzyme A to split off the carboxyl-terminal
two-carbon fragment of the original fatty acid as
acetyl-CoA.
The four β-Oxidation steps are repeated to yield Acetyl-CoA
& ATP
• One molecule of acetyl-CoA, two pairs of electrons
(H+) are removed from the long-chain fatty acyl-CoA,
shortening it by 2 carbon atoms.
Example: Palmitate
• Yields 106 molecules of ATP
• Cn-acyl CoA + FAD + NAD+ + H2O + CoA = Cn-2-acyl CoA
+ FADH2 + NADH + acetyl CoA + H+
• Cn-acyl CoA + FAD + NAD+ + H2O + CoA =
Cn-2-acyl CoA + FADH2 + NADH + acetyl CoA + H+
• Palmitoyl CoA + 7FAD + 7NAD+ + 7CoA + 7H2O =
8 Acetyl CoA + 7FADH2 + 7NADH + 7H+
Oxidation of Unsaturated Fatty Acids requires two additional
reactions
• The cis configuration cannot be acted upon by enoyl-
CoA hydratase, the enzyme catalyzing the addition of
28
H2O to the trans double bond of the Δ2-enoyl-CoA
hydratase generated during β oxidation.
Oleate
• abundant 18-carbon monounsaturated fatty acids
with a cis double bond between C-9 and C-10 (Δ9)
• Oleate is converted to oleoyl-CoA and, like the
saturated fatty acids, enters the mitochondrial matrix
via the carnitine shuttle.
• Oleoyl-CoA then undergoes three passes through the
fatty acid oxidation cycle to yield 3 molecules of Acetyl-
CoA & the coenzyme A ester of Δ3, 12-carbon
unsaturated fatty acid, cis-Δ3-dodecenoyl-CoA
• this product cannot serve as substrate for enoyl-CoA
hydratase (only on trans double bond)
• Δ2-enoyl-CoA isomerase, enzymes that isomerizes the
cis-Δ3-enoyl-CoA to the trans-Δ2-enoyl-CoA, which is
converted by enoyl-CoA hydratase into L-β-
hydroxyacyl-CoA.
• Latter undergoes 4 more passes through β-oxidation
pathway to yield 5 molecules of Acetyl-CoA.
β-oxidation (Even)
Example: Lauric acid (C20:0)
1.) Determine the no. of Acetyl CoA from the fatty
acid molecule
𝑵𝑵𝑵𝑵.𝒐𝒐𝒐𝒐 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂
Formula:
𝟐𝟐
Then multiply by 10 to convert into ATP
2.) Determine the no. of steps of Fatty acids that
would undergo
𝑁𝑁𝑁𝑁.𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
No. of steps = −1
2
No. of steps multiply by 4 = no. of ATP generated
3.) Total ATPs – Minus 2 ATP for the activation
• No. Of Acetyl CoA = 6 x 10 = 60
12
• No. of steps = − 1 = 5 x 4 = 20
2
• 60 ATP + 20 ATP – 2= 78 ATPs
 β-oxidation (Odd) • In fasting or diabetes, oxaloacetate is consumed to
form glucose by the gluconeogenic pathway & hence
1.) Determine the no. Acetyl CoA from the fatty acid is unavailable for condensation with Acetyl CoA.
molecule • Acetyl CoA is diverted to the formation of
acetoacetate & D-3-hydroxybutyrate.
𝑁𝑁𝑁𝑁.𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
Formula: • Also known as ketone bodies
2
Then multiply by 10 to convert to ATP, 1 propionyl CoA • Acetoacetate is formed from Acetyl CoA in 3 steps.
produces 5 ATP Two molecules of acetyl CoA condense to form
acetoacetyl CoA.
2.) Determine the no. of steps of fatty acid that undergo
beta oxidation Enzyme: Thiolase

• Acetoacetyl then reacts with acetyl CoA & water to

𝑁𝑁𝑁𝑁.𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
No. of steps = −1 give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) & CoA
2

No. of steps multiply by 4 = no. of ATP generated

3.) Total ATPs – Minus 3 ATP for the activation

 No. acetyl CoA
 No. of steps
 No. of propionyl CoA

Fatty acids are also oxidized in peroxisomes

• Although most fatty acids oxidation takes place in

mitochondria, oxidation of long chain & branched
takes place in cellular organelles (Peroxisomes). Ketone bodies are a major fuel in some tissues
• Serves to shorten very long fatty chains (C26) to make
• The major site of the production of acetoacetate and
them better substrates of beta-oxidation in
3-hydroxybutyrate is the liver.
mitochondria.
• In peroxisomes, acyl CoA dehydrogenase, a
• These molecules diffuse from liver mitochondria into
flavoprotein, transfer electrons from the substrate to
the blood and are transported to other tissues such as
FADH2 & then O2 to yield H2O2.
heart & kidney.
• Peroxisomes contain high concentrations of the
enzyme catalase to degrade H2O2 into water and O2.
•
Ketogenesis
• Acetoacetate & 3-hydroxybutyrate are normal fuels of
respiration and are quantitatively important as
sources of energy.

• Acetoacetate is converted into acetyl CoA in two steps

(1) Acetoacetate is activated by the transfer of CoA

from succinyl CoA in a reaction catalyzed by a
specific CoA transferase.
Ketone bodies are formed from acetyl CoA when fat
(2) Acetoacetyl CoA is cleaved by thiolase to yield
breakdown predominates
two molecules of acetyl CoA, which then enter
the TCA cycle.
• If carbohydrate is unavailable or improperly utilized,
(3) The liver has acetoacetate available to supply
the concentration of oxaloacetate is lowered & acetyl
other organs because it lacks this particular CoA
CoA cannot enter the TCA.
transferase.

29
 • 3-Hydroxybutyrate requires an additional step to yield - Increased β-oxidation results in more production
acetyl CoA of ATP through citric acid cycle in liver.
- Increased availability of acetyl CoA for
(1) Oxidized to produce acetoacetate ketogenesis.

Disease Enzyme Clinical Manifestation

Lipid anabolism
deficient/Cause
Chanarin- Missing Dry skin (ichthyosis),
Dorfman coactivator of enlarged liver, muscle • Construction or synthesis of structural and functional
syndrome ATGL weakness & mild lipids
cognitive disability.
• Responsible for development of cell membranes in
Medium-chain Medium-chain Unable to break down
mostly animals
acyl-CoA acyl-CoA certain fats. C
dehydrogenase dehydrogenase • Lipogenesis – a process of synthesizing fats
(MCAD) • Majority of lipids found in the human body are
deficiency obtained from triglycerides and fatty acids
Muscle weakness Carnitine Muscle, heart &
deficiency kidney are primarily
Interaction Between Lipids
impaired. Muscle
weakness during
prolonged exercise.
Zellweger Defect in the Liver, kidney, and
Syndrome import of muscle abnormalities.
enzymes into Can led to death.
the
peroxisomes

General Reaction Scheme for Triglycerides reacting with fatty

acids

Note: High levels of acetoacetate in the blood signify
an abundance of acetyl units & lead to a decrease in
the rate of lipolysis in adipose tissue.
High blood levels of ketone bodies can also be life
threatening.
• Saturated Fatty Acids – only produce single bonds
• Monounsaturated Fatty Acids – only has one double
bond
• Polyunsaturated Fatty Acids – have two or more
double bonds

Regulation of ketogenesis

• Substrate availability
- Increased ketogenesis occurs when there is
excessive availability of fatty acids for oxidation.
Thus, increased ketogenesis occurs during
“Saturated Fatty Acids” “Cis double bond in
diabetes milletus or starvation
Unsaturated fatty
• Regulation of β-Oxidation Acids”
- Increased glucagon & decrease insulin in fasting
result in inhibition of acetyl CoA carboxylase.
- This decrease in malonyl CoA
• Availability of ATP
30
 • Regularly distributed Fatty Acids have carbon tails
interact through relatively strong London Forces
• Irregularly shaped Fatty Acids interact less efficiently
(due to their shape) resulting in weaker London forces
Lipid Mechanism
Pentose Phosphate Pathway
Saponification of Fatty Acids with Base forming Cation Salts
• Occurs mainly in Cytoplasm
• Incorporated with Carbon Atoms from Acetyl CoA
into the growing fatty acid Chain
Biological representation of cell membranes from Fatty Acids
• These Fatty acid salts will form with the influence of
opposing charges of different molecules called
micelles
• In terms of biological aspects, cell membranes are
responsible in protecting cells from foreign
microorganism
Lipid Metabolism
Visual representation of Fatty Acid Metabolic Pathway
31
• Fatty Acids are responsible for Main Energy
Metabolic Processes
• Excess fats are stored in special type of tissues called
Adipose
• These type of tissues are stored for future energy
conversion, one example can be hibernation of
specific types of animals
Pentose Phosphate Pathway general pathway
• Functions of this alternative pathway
o Synthesis of the coenzyme NADPH needed in
lipid biosynthesis
o From glycolysis, glucose is used to produce
NADPH
o NADPH is used for Anabolic Processes
requiring electrons
Fatty Acid Synthesis Metabolic pathway
• Acetyl CoA (water Soluble) is converted into Citrate
(1st reaction of TCA cycle)
• In to the Cytosol, citrate undergo reverse reaction
producing oxaloacetate and Acetyl CoA
• If the condition is favoured, FA synthesis begins
• Acetyl CoA is then Activated which will be needed for
the synthesis of FA chain via Addition reaction
Activation of Acetyl CoA Fatty Acid Synthesis

• Condensation

Initiation of Fatty Acid Synthesis

• Formation of malonyl CoA through irreversible

carboxylation of acetyl CoA
• A CO2 is attached to acetyl CoA producing 3C molecule
• Hydrolysis of ATP supply the energy needed to attach
the CO2
• Malonyl-CoA is an Activated form of Acetyl CoA used for
FA biosynthesis
•

Nucleophilic attack breaks the thioester bond

• Decarboxylation reaction involves cleavage of

high energy thioester bond – which supply
enough energy to drives the whole reaction
forward
o The acetyl group moves from cysteine
residue onto malonate group on ACP

• Reduction by NADPH (first part)

Regulatory step for interconversion in FA synthesis

• Malonyl CoA inhibits CAT1 to prevent the entry of fatty

acid into the mitochondria
• Acetyl transacylase and malonyl transacylase catalyze
Acetyl CoA and Malonyl CoA
• Propionyl CoA is binded for odd chain synthesis

Reduction to stabilize redox reaction

o Acetoacetyl is transformed to D-3-

hydroxybutyl by converting the carbonyl
group into the alcohol group
o Reducing agent: NADPH + H+ → NADP+
o Catalyzing enzyme: β-ketoacyl ACP
reductase

32
• Dehydration
Dehydration to form double bond by removing water
o Removal of hydroxyl group as H2O and
formation of C=C
o Catalyze by 3-hydroxyacyl ACP
dehydrase
• Reduction by NADPH (second part)
Reduction by NADPH is needed to end for possible
recycle process
o Butyryl group is generated on ACP
o NADPH reduced the C=C because the
ultimate FA chain produced is a
saturated FA
o This completes the first elongation
process containing 4C atoms
o This whole cycle takes place 7 times in
total to generate a 16C palmitate
• Restarting the Cycle
33
o From the 16C palmitate, 14C are from
Malonyl-CoA
o The other 2 C are from Acetyl CoA
Lipid-related Disease
o GAUCHER DISEASE
- caused by the deficiency of Glucocerebrosidas,
- Fatty acid Enzyme deficient used for FA synthesis
- can affect most parts of Kidneys, Livers, Blood-
control flow, lungs and Brain
Three common Types:
o Type 1 (non neuronopathic type)
 Symptoms may begin early in life or in
adulthood
 May cause enlargement of vital organs like
liver or spleen which causes rupture
o Type 2 ( acute infantile neuropathic type)
 Typically begins from 3 months of birth
 May include symptoms extensive and
progressive brain damage, seizures and
other brain-related disorders
o Type 3 (chronic neuropathic form)
 can begin at any time between childhood or
adulthood
 characterize as slowly progressive, but
milder neurologic disorders
 can have the same effects to type 1 and type
2
Questions:
1.) The best storage form. Compare the ATP yield from
the complete oxidation of glucose, a six- carbon
carbohydrate, and hexanoic acid, a six-carbon fatty
acid. Hexanoic acid is also called caprioic acid and is
responsible for the “aroma” of goats. Why are fats
better fuels than carbohydrates?
2.) What are the coenzymes used in synthesis of Lipids?
3.) Generous, but not to a fault. Liver is the primary site of
ketone-body synthesis. However, ketone bodies are
not used by the liver but are released for other tissues
to use. The liver does gain energy in the process of
synthesizing and releasing ketone bodies. Calculate
the number of molecules of ATP generated by the liver
in the conversion of palmitate, a C16 fatty acid, into
acetoacetate.
4.) What are the regulatory steps in FA synthesis?
• The eight molecules of acetyl CoA combine to
5.) Counting ATPs 2. How much energy is attained with
the complete oxidation of the ketone body D-3-
hydroxybutyrate?
6.) Why do we need NADPH for reduction?
7.) Counting ATPs 1. What is the ATP yield for the
complete oxidation of C17 (heptadecanoic) fatty
acid? Assume that the propionyl CoA ultimately yields
oxaloacetate in the citric acid cycle.
8.) How many ATP’s are consumed in FA synthesis?
• NADH produced with the oxidation to
9.) Comparing yields. Compare the ATP yields from
palmitic acid and palmitoleic acid
10.) Why is Malonyl CoA is activated before FA synthesis?
ANSWER:
• Palmitic acid yields 106 molecules of ATP.
• Keep in mind that, in the citric acid cycle, 1
molecule of FADH 2 yields 1.5 ATP, 1 molecule
of NADH yields 2.5 ATP, and 1 molecule of acetyl
CoA yields 10 ATP. Two molecules of ATP are
produced when glucose is degraded to 2
molecules of pyruvate. Two molecules of NADH
also are produced, but the electrons are
transferred to FADH 2 to enter the electron
transport chain. Each molecule of FADH 2 can
generate 1.5 ATP. Each molecule of pyruvate
will produce 1 molecule of NADH. Each molecule
of acetyl CoA generates 3 molecules of NADH, 1
molecule of FADH 2 ,and 1 molecule of ATP. So,
we have a total of 10 ATP per acetyl CoA, or 20
for the 2 molecules of acetyl CoA. The total for
glucose is 30 ATP. Now, what about hexanoic
acid? Caprioic acid is activated to caprioic CoA at
the expense of 2 ATP, and so we are 2 ATP in the
hole. The first cycle of b oxidation generates 1
FADH 2 , 1 NADH, and 1 acetyl CoA.After the
acetyl CoA has been run through the citric acid
cycle, this step will have generated a total of 14
34
ATP. The second cycle of b oxidation generates 1
FADH 2 and 1 NADH but 2 acetyl CoA. After the
acetyl CoA has been run through the citric acid
cycle, this step will have generated a total of 24
ATP. The total is 36 ATP. Thus, the foul-smelling
caprioic acid has a net yield of 36 ATP. So on a
per carbon basis, this fat yields 20% more ATP
than does glucose, a manifestation of the fact
that fats are more reduced than carbohydrates.
form four molecules of acetoacetate for release
into the blood, and so they do not contribute to
the energy yield in the liver. However, the FADH
2 and NADH generated in the preparation of
acetyl CoA can be processed by oxidative
phosphorylation to yield ATP.
1.5 ATP/FADH2 x 7 = 10.5 ATP
2.5 ATP/NADH x 7 = 17.5 ATP
The equivalent of 2 ATP were used to form
palmitoyl CoA. Thus, 26 ATP were generated for
use by the liver.
acetoacetate 5 2.5 ATP. Acetoacetate is
converted into acetoacetyl CoA. Two molecules
of acetyl CoA result from the hydrolysis of
acetoacetyl CoA, each worth 10 ATP when
processed by the citric acid cycle. Total ATP yield
is 22.5.
Palmitoleic acid has a double bond between
carbons C-9 and C-10. When palmitoleic acid is
processed in b oxidation, one of the oxidation
steps (to introduce a double bond before the
addition of water) will not take place, because
a double bond already exists. Thus, FADH 2 will
not be generated, and palmitoleic acid will yield
1.5 fewer molecules of ATP than palmitic acid, for
a total of 104.5 molecules of ATP.
Summary

 Lipids are fats that are either absorbed from food or

synthesized by the liver. Triglycerides (TG’s) and
cholesterol contribute most to disease, although all
lipids are physiologically important.
 All lipids are hydrophobic and mostly insoluble in
blood, so they are classified by size and density
(defined as ratio of lipid to protein) and are
important because of high levels of Low Density
Lipoprotein (LDL) and low levels of High Density
Lipoproteins (HDL) are major risk factors for various
Heart diseases.
 Cholesterol is a ubiquitous constituent of cell
membranes. Steroids, bile acids, and signaling
molecules.
 Triglycerides primary store energy in adipocytes and
muscle cells
 Lipoprotein are hydrophilic, spherical structures that
possess surface proteins (apoproteins or
apolipoproteins) that are cofactors and ligands for
lipid processing enzyme

35
 Adamson University May 24, 2019
SY 2018 – 2019 Proteins and Enzymes ABLAY, Beatriz G.
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. MACARAIG, Marty L.

Outline of the Lecture

I. Introduction to Proteins
II. Amino Acids: Building Blocks of Proteins
III. The Three-Dimensional Structure of Proteins
IV. Isolation and Purification Techniques of Proteins
V. Enzymes

Introduction to Proteins
Proteins (Greek proteios, “primary” or “of first importance”) are
biochemical molecules consisting of polypeptides joined by
peptide bonds between the amino and carboxyl groups of amino
acid residues.

Proteins perform a number of vital functions:

• Enzymes are proteins that act as biochemical
catalysts. Fig. 2. Classification of Amino Acids according to its Polarity
• Many proteins have structural or mechanical
functions (e.g. actin and myosin in muscles).  Based on essentiality
• Proteins are important in cell signaling, immune Essential amino acids are the amino acids which you
responses, cell adhesion, and the cell cycle. need through your diet because your body cannot
• Proteins are a necessary component in animal make them.
diets.
Non-essential amino acids are the amino acids which
Amino Acids: Building Blocks of Proteins are not an essential part of your diet because they can
Amino Acids contain both a carboxylic group and an amino be synthesized by your body.
group. Amino acids that have an amino group bonded directly
to the alpha-carbon are referred to as alpha amino acids. Table 1. List of Essential and Non-essential Amino Acids
Essential Non-Essential
Every alpha amino acid has a carbon atom, called an alpha Histidine Alanine
carbon, Cα ; bonded to a carboxylic acid, –COOH group; an Isoleucine Arginine
amino, –NH2 group; a hydrogen atom; and an R group that is Leucine Aspargine
unique for every amino acid. Methionine Aspartate
Phenylalanine Cystine
Threonine Glutamic acid
Tryptophan Glycine
Proline
Serine
Tyrosine

 Acid-Base Properties of Amino Acids

Amino acids contain both an acidic group and an alkaline group

Fig. 1. General Structure of Amino Acid

 Classification of Amino Acids

 Based on Polarity
Fig. 3 The Acidic and Basic Group of Amino Acid
The nature of the R group of amino acid is the basis of its
polarity.

• In neutral aqueous solution

36
Carboxyl groups donate H+, producing a negative charge ions.
―COOH- + H+

Amino groups accept H+, producing a negatively charge ions.

―NH2 + H+ → ―NH3+

An internal acid-base reaction occurs in the same amino acid

which produces zwitterion. Fig. 6. Structural Changes of Zwitterion when the pH of the
system is basic.

In a solution, amino acids have three different forms. It is

induced by shift in the equilibrium caused by pH changes.

Three forms:
• Zwitterion
• Positive ion
Fig. 4. Zwitterion
• Negative ion
Isoelectric point is the characteristics pH, at which a specific
amino acid exists as a zwitterion and its net charge is zero.
Therefore, at isoelectric point, the amino acids are not
attracted toward electric field. Different amino acids have
different isoelectric points and it is dependent on the structure
of the amino acid.

Fig. 7. Structural Changes of Amino Acid, Alanine when the pH

is altered.

 Cysteine: A Unique Amino Acid

Cysteine is the only standard amino acid containing a sulfhydryl

group –SH(thiol group). It readily oxidizes & forms a disulfide
linkage with another cysteine. The two cysteine residues linked
via a disulfide bond help maintain protein structure, stability &
function. Disulfide bonds contributes to tertiary, quaternary
protein structure.

Fig. 5. Isoelectric points for 20 Amino Acids Commonly Found

Fig. 8. Two Cysteine Residues Forming a Disulfide Bond
in Proteins
 Amino Acids forming Proteins: Peptide Bond
Zwitterion structure changes when pH is altered
Amino acids are linked together in proteins by peptide bonds.
• In a solution more alkaline than the isoelectric point
Covalent Amide bond found between amino acids in a peptide
Zwitterion donates a H+ from both amino and
chain.
carboxyl groups and forms a negatively charged ion.
Peptide bond formation involves carboxyl group (–COOH ) of
one amino acid interacts with the amine group (–NH2) of
another amino acid
• Produces an Amide + a molecule of H2O

37
 • Quaternary Structure (4ᵒ): Overall arrangement
of protein structure involving multiple peptide
chains.

 Primary Structure (°1) of Proteins

Fig. 9. Two Amino Acid Residues Forming a Peptide Bond
Primary structure is the sequence of amino acids in a protein
chain.
 Amino Acid Chain: Peptides

Peptide is unbranched chain of amino acids held together by
peptide bonds.
A peptide chain has 2 different ends:
• The end carrying a free –NH3+is called the N-
terminal end
• The end carrying a free –COO-is called the C-
terminal end
• Involves the number, type & order of attachment of the
amino acids.
• Every protein has a different amino acid sequence.
Insulin was the first protein to have its primary structure
determined.
• 51 amino acids in 2 chains, linked via disulfide bonds.
• Chain A (21 AAs) & Chain B (30 AAs)

By convention, the sequence of amino acids in a peptide is

always written with N-terminal on the left & the C-terminal on
the right.

Different Kinds of Peptides

• Dipeptide
2 amino acids linked via peptide bond

• Tripeptide
3 amino acids linked via peptide bond Fig. 10. Primary Structure of Insulin

• Oligopeptide  Secondary Structure (°2) of Proteins

10 to 20 amino acids linked via peptide bond
• Polypeptide
Long unbranched chain of 20+ amino acids linked
via peptide bond
The Three-Dimensional Structure of Proteins
General Structure of Proteins is the three-dimensional
arrangement of atoms in an amino acid-chain molecule.
Proteins are polymers specifically polypeptides formed from
sequences of amino acids, the monomers of the polymer.
The 3-D structure of all proteins both monomeric or multimeric,
is more complex than that of carbohydrates or lipids.
Secondary structure is the arrangement of the primary Protein
Structure in space
• Formed by hydrogen bonds between backbone
atoms. Hydrogen bonds form between the oxygen of
the C=O of one amino acid & the H of the N–H of
another amino acid within the protein
2 Types of Structure:
1. Alpha-helix
 Shape of a coiled spring (Helix)
 Hydrogen bonds are formed between C=O and NH of
the 4th amino acid down the same protein chain
 The R-groups stay outside of the helix

4 Levels of Protein Structure:

• Primary Structure (1ᵒ): amino acid sequence

• Secondary Structure (2ᵒ): α-helices, β-strands,

random coil (absence of 2ᵒ structure)

• Tertiary Structure (3ᵒ): Overall arrangement of

protein structure within one peptide chain

Fig. 11. Illustration of Alpha-helix Structure of Proteins

38
2. Beta-pleated sheet
 Formed between 2 protein chain segments side by
side.
 Linked by hydrogen bonds.
 The R-groups are above & below the sheet.
Fig. 12. Illustration of Beta-pleated Sheet Structure of Proteins
 Tertiary Structure (°3) Structure of Proteins
Tertiary protein structure is formed by interactions between
the side chains (R-groups) of the amino acids within a protein.
Result in a more complex 3-D arrangement of the protein in
space.
4 Types of Interactions:
1. Disulfide bonds: The strongest interactions (covalent
bond)
• Between two cysteine’s
2. Electrostatic interactions (Salt Bridges)
• Interactions between an acidic & a basic R-group
3. Hydrogen bonds: between polar R-groups
4. Hydrophobic attractions: between non-polar R-groups
Fig. 13. Interactions in Tertiary Structure
 Quaternary Structure (°4) Structure of Proteins
Highest level of protein organization found only in multimeric
proteins that have 2 or more polypeptide chains (subunits) in
their structure.
• Subunits are held together by the same interactions
as tertiary structure:
Hydrogen bonds, disulfide bonds, hydrophobic &
electrostatic interactions
39
• 4ᵒ structure easily disrupted by very small changes in
cellular conditions. Protein chains fall apart, resulting
in a temporary loss of protein activity. When normal
conditions are restored the multimer automatically
reforms & regains its function.
Isolation and Purification Techniques of Proteins
A major portion of most biochemical investigations involves the
purification of the materials under consideration because these
substances must be relatively free of contaminants if they are
to be properly characterized.
 Protein Isolation
Isolating the protein to be studied comprehensively.
Four Major Steps of Protein Isolation
1. Selection of a Protein Source
2. Methods of Solubilization
3. Stabilization of Proteins
4. Assay of Proteins
5. General Strategy of Protein Purification
 Solubilities of Proteins
Proteins are conveniently purified on a large scale by a
fractional precipitation process called salting out, in which
protein solubilities are varied by changing the salt
concentration or pH.
 Chromatographic Separations
• Ion exchange chromatography employs support
materials such as cellulose or cross-linked dextran
gels. Separations are based on differential
electrostatic interactions between charged groups
on the ion exchange materials and those on the
substances being separated.
• Molecules may be located through their UV
absorbance, fluorescence, radioactivity, or enzymatic
activity.
• In gel filtration chromatography, molecules are
separated according to their size and shape through
the use of cross-linked dextran, polyacrylamide, or
agarose beads that have pores of molecular
dimensions. A calibrated gel filtration column can be
used to estimate the molecular masses of
macromolecules.
• Affinity chromatography separates biomolecules
according to their unique biochemical abilities to
bind other molecules specifically.
• High-performance liquid chromatography (HPLC)
utilizes any of the foregoing separation techniques
but uses high resolution chromatographic materials,
high solvent pressures, and automatic solvent mixing
and monitoring systems so as to obtain much greater
 degrees of separation than are achieved with the DNA from double-stranded DNA. Polyacrylamide or
more conventional chromatographic procedures. agarose gel electrophoresis separates DNA largely on the
• Adsorption chromatography, thin layer basis of size. Very large DNAs can be separated by pulsed-
chromatography (TLC), reverse-phase field gel electrophoresis (PFGE) on agarose gels.DNAs may
chromatography (RPC), hydrophobic interaction be fractionated according to their base composition by
chromatography (HIC), and metal chelate affinity CsCl density gradient ultracentrifugation. Different species
chromatography also have valuable biochemical of RNA can be separated by zonal ultracentrifugation
applications. through a sucrose gradient.

 Electrophoresis Enzymes
In electrophoresis, charged molecules are separated Enzymes are a protein with catalytic properties due to its power
according to their rates of migration in an electric field on of specific activation. It is the most remarkable and highly
a solid support such as paper, cellulose acetate, crosslinked specialized protein.
polyacrylamide, or agarose. • Catalyze hundreds of stepwise reactions in
biological systems.
• Regulate many different metabolic activities
• Gel electrophoresis employs a cross-linked
necessary to sustain life.
polyacrylamide or agarose gel support, so that
molecules are separated according to size by gel  Mechanism of Enzymes
filtration as well as according to charge. The Enzymes provide a specific environment within which a given
separated molecules may be visualized by means reaction is energetically more favorable.
of stains, autoradiography, or immunoblotting. • Active site confinement of a pocket on the enzymes
The anionic detergent sodium dodecyl sulfate where an enzyme-catalyzed reaction occurs.
(SDS) denatures proteins and uniformly coats • Substrate molecule that is bound by the active site and
them so as to give most proteins a similar charge acted upon by the enzyme.
density and shape. SDS–PAGE may be used to
estimate macromolecular masses. In isoelectric
focusing (IEF), macromolecules are immersed in a
stable pH gradient and subjected to an electric
field that causes them to migrate to their
isoelectric positions. In capillary electrophoresis,
the use of thin capillary tubes and high electric
fields permits rapid and highly resolved
separations of small amounts of material.

 Ultracentrifugation Fig. 14. Illustration of Mechanism of Enzymes

In ultracentrifugation, molecules are separated by
subjecting them to gravitational fields large enough to Classification of Enzymes
counteract diffusional forces. Molecules may be separated,
and their molecular masses estimated from their rates of ENZYMES TYPE OF REACTION CATALYZED
sedimentation through a solvent or a preformed gradient Oxidoreductases Oxidation-reduction reactions
of an inert low molecular mass material such as sucrose. Transferases Transfer of functional groups
Hydrolases Hydrolysis reaction
Alternately, molecules may be separated according to their Lyases Group elimination to form double
buoyant densities in a solution with a density gradient of a bond
dense, fast-diffusing substance such as CsCl. The deviation Isomerases Isomerization reaction
of a molecule’s frictional ratio from unity is indicative of its Ligases Bond formation couples with ATP
hydrolysis
degrees of solvation and elongation.

 Nucleic Acid Fractionation

Nucleic acids can be fractionated by many of the
techniques that are used to separate proteins.
Hydroxyapatite chromatography separates single stranded

40
 Summary
I. Introduction to Proteins
Proteins are biochemical molecules consisting of
polypeptides joined by peptide bonds between the amino and
carboxyl groups of amino acid residues. They perform vital
functions in the body such as catalyzing biochemical reactions
and plays an important role in the cell cycle.

II. Amino Acids: Building Blocks of Proteins

Amino Acids are the building blocks of proteins and
contain both a carboxylic group and an amino group. They have
both an acidic group and an alkaline group. Amino acids can be
classified according to their polarity and essentiality.
Isoelectric point is the characteristics pH, at which a
specific amino acid exists as a zwitterion and its net charge is
zero. Different amino acids have different isoelectric points and
it is dependent on the structure of the amino acid.

III. The Three-Dimensional Structure of Proteins

The general structure of proteins is the three-
dimensional arrangement of atoms in an amino acid-chain
molecule. The levels of protein structure can be classified as
primary, secondary, tertiary and quaternary.
Primary structure is the sequence of amino acids in a
protein chain. Secondary structure is the arrangement of the
primary protein structure in space. Tertiary protein structure is
formed by interactions between the side chains (R-groups) of
the amino acids within a protein, resulting in a more complex
3-D arrangement in space. Quaternary protein structure is
found only in multimeric proteins that have 2 or more
polypeptide chains (subunits) in their structure.

IV. Isolation and Purification Techniques of Proteins

Proteins can be isolated and purified to study them
comprehensively. They can be isolated depending on their
solubility and through instrumental methods including
chromatography, electrophoresis and ultracentrifugation.

V. Enzymes
Enzymes are proteins with catalytic properties due to
their power of specific activation. The catalyze hundreds of
stepwise reactions in biological systems and regulate different
metabolic activities necessary to sustain life. Enzymes can be
classified according to the types of reaction they catalyze.

41
 Adamson University
SY 2018 – 2019 Enzymes May 24, 2019.
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Musor & Omos

Outline of the Lecture Coenzyme Chemical Groups Dietary Precursor

I. An Introduction to Enzymes Transferred in
II. How does enzyme work? Mammals
III. FACTORS AFFECTING REACTION VELOCITY Biocytin CO2 Biotin
IV. MICHAELIS-MENTEN EQUATION Lipoate Electron and Not required
V. INHIBITION OF ENZYME ACTIVITY Acyl Groups in diet
VI. REGULATION OF ENZYME ACTIVITY Coenzyme A Acyl Groups Panthothenic and
VII. ENZYMES IN CLINICAL DIAGNOSIS other
compounds
An Introduction to Enzymes Tetrahydrofolate One-carbon Folate
Life depends on powerful and specific catalysts such as groups
enzymes, where every biochemical reaction depends. All Thiamine Aldehydes Thiamine
enzymes are protein- except for catalytic RNA molecules. The pyprophosphate
catalytic activity of an enzyme depends solely on the protein Flavin Adenine Electrons Riboflavin
conformation. If an enzyme is denatured or dissociated into dinucleotide
subunits or broken down into amino acids, catalytic activity is
Pyridoxal Amino groups Pyrodoxine
typically lost. Their structures are also essential to their catalytic
phosphate
activity. Properties:
Table 2: Some Coenzymes that serve as Transient Carriers of
 Primary
Specific Atoms
 Secondary
 Tertiary How does enzyme work?
 Molecular Weights ranging from 12,000 to more An enzyme circumvents the reactions that need catalysts to
than 1 million. occur by providing a specific environment which a given
 Cofactor for an additional chemical component (see reaction can occur more rapidly. The enzyme-catalyzed takes
table 1). place within the confines of a pocket on enzyme called active
site. The molecule that is bound in active site is called substrate.
 Coenzyme a complex-organic or metalloorganic
molecule that act as a carrier for specific function.
Ions Enzymes

Cu2+ Cytochrome oxidase

Fe2+ or Fe3+ Cytochrome oxidase,

catalase, peroxidase
K+ Pyruvate Kinase

Mg2+ Hexokinase, glucose-6-

phosphate, pyruvate
kinase
Mn2+ Arginase, ribonucleotide
reductase
Figure 1: Binding of a substrate to an enzyme at the active
2+ site.
Ni Urease

Zn2+ Carbonic anhydrase,

alcohol dehydrogenase
Table 1: Inorganic Ions as Cofactors for Enzymes

42
A simple enzyme reaction that might be write as - The rate of an enzyme-catalyzed reaction increases with
substrate concentration until a maximal velocity (Vmax) is
reached.

- The leveling off of the reaction rate at high substrate

concentrations reflects the saturation with substrate of all
available binding sites on the enzyme molecules present.
(Figure 1.)

 Hyperbolic shape of the enzyme kinetics curve

- Most enzymes show Michaelis-Menten kinetics, in which the

plot of initial reaction velocity (Vo) against substrate
where E, S, and P represent the enzyme, substrate and P
concentration ([S]) is hyperbolic.
represent the enzyme, substrate, and product; ES and EP are
transient complexes of enzyme with substrate and with the - In contrast, allosteric enzymes do not follow Michaelis-
product. The enzyme doesn't affect the equilibria of a reaction Menten kinetics and show a sigmoidal curve that is similar in
but only the rate of the reaction. shape to the oxygen dissociation curve of hemoglobin.
(Figure 3)
In order for a reaction to occur, the molecules present must
overcome must have sufficient energy to overcome the
energy barrier between them which is called the activation
energy. There are two kinds of model to explain how enzymes
works.

1. Lock and Key Model

This is the simplest model to represent how a substrate simole

fits to the active site to form reaction intermediate. Figure 3: Effect of substrate concentration on reaction velocity

Figure 2: Lock and Key Model B. Temperature

1. Induced Fit Model
In this model, the enzyme molecule changes shape as the
substrate molecule gets closer. The change in shape is induced
by the approaching substrate molecule.
FACTORS AFFECTING REACTION VELOCITY
- The reaction velocity increases with temperature until a peak
velocity is reached.
- Further elevation of the temperature results in a decrease in
reaction velocity as a result of temperature induced
denaturation of the enzyme. (Figure 4)

OVERVIEW :

Different enzymes show different responses to changes in

substrate concentration, temperature, and ph. This section
describes factors that influence the reaction velocity of
enzymes.

A. Substrate concentration

 Maximal Velocity
Figure 4: Effects on temperature
- The rate or velocity of a reaction (v) is the number of
substrate molecules converted to product per unit time;
velocity is usually expressed as μmol of product formed per
minute. C. pH

 Effect of pH on the ionization of the active site

43
- The concentration of H+ affects reaction velocity in several
ways. First, the catalytic process usually requires that the
enzyme and substrate have specific chemical groups in
either an ionized or un-ionized state in order to interact.
(Figure 5)
 Effect of pH on enzyme denaturation
- Extremes of pH can also lead to denaturation of the enzyme,
because the structure of the catalytically active protein
molecule depends on the ionic character of the amino acid
side chains.
 The pH optimum varies for different enzymes
- The pH at which maximal enzyme activity is achieved is
different for different enzymes, and often reflects the [H+] at
which the enzyme functions in the body.
Figure 5. Effects on pH w/ different Enzymes
MICHAELIS-MENTEN EQUATION
A. Reaction model
- In this model, the enzyme reversibly combines with its
substrate to form an ES complex that subsequently yields
product, regenerating the free enzyme.
Represented model below shows one substrate molecule:
A. Michaelis-Menten equation
- The Michaelis-Menten equation describes how reaction
velocity varies with substrate concentration:
44
- Several assumptions that are made in deriving the Michaelis-
Menten:
 Relative concentrations of E and S:
- The concentration of substrate ([S]) is much greater than the
concentration of enzyme ([E]), so that the percentage of
total substrate bound by the enzyme at any one time is
small.
 Steady-state assumption
- [ES] does not change with time (the steady-state
assumption), that is, the rate of formation of ES is equal to
that of the breakdown of ES (to E + S and to E + P).
- In general, an intermediate in a series of reactions is said to
be in steady state when its rate of synthesis is equal to its
rate of degradation.
 Initial velocity
- Initial reaction velocities (vo) are used in the analysis of
enzyme reactions.
- This means that the rate of the reaction is measured as soon
as enzyme and substrate are mixed.
- At that time, the concentration of product is very small and,
therefore, the rate of the back reaction from [P] to
[S] can be ignored.
Figure 6. Effects of Substrate concentration on reactions for
two enzymes
 B. Important conclusions about Michaelis- Menten
kinetics
 Characteristics of [Km]
- [Km] the Michaelis constant is characteristic of an enzyme
and its particular substrate and reflects the affinity of the
enzyme for that substrate.
- [Km] is numerically equal to the substrate concentration at
which the reaction velocity is equal to 1⁄2Vmax.
- [Km] does not vary with the concentration of enzyme.
A. Lineweaver-Burk plot
- The Lineweaver-Burk plot (also called a double reciprocal
plot) can be used to calculate Km and Vmax, as well as to
determine the mechanism of action of enzyme inhibitors.
(Figure 8)
1. The equation describing the Lineweaver-Burk plot is.

a) Small [Km]
Where the intercept on the x-axis is equal to -1/Km, and the
- A numerically small (low) Km reflects a high affinity of the
intercept on the y-axis is equal to 1/Vmax.
enzyme for substrate, because a low concentration of
substrate is needed to half-saturate the enzyme that is, to
reach a velocity that is 1⁄2Vmax.

b) Large [Km]
- A numerically large (high) Km reflects a low affinity of
enzyme for substrate because a high concentration of
substrate is needed to half-saturate the enzyme.
-
 Relationship of velocity to enzyme concentration
- The rate of the reaction is directly proportional to the
enzyme concentration at all substrate concentrations. Figure 8. Lineweaver plot
-
 Order of reaction
- When [S] is much less than Km, the velocity of the reaction is INHIBITION OF ENZYME ACTIVITY
approximately proportional to the substrate concentration.
- The rate of reaction is then said to be first order with respect - Any substance that can diminish the velocity of an enzyme-
to substrate. When [S] is much greater than Km, the velocity catalyzed reaction is called an inhibitor.
is constant and equal to Vmax. - In general, irreversible inhibitors bind to enzymes through
- The rate of reaction is then independent of substrate covalent bonds.
concentration and is said to be zero order with respect to - Reversible inhibitors typically bind to enzymes through non-
substrate concentration. covalent bonds, thus dilution of the enzyme inhibitor complex
results in dissociation of the reversibly bound inhibitor, and
recovery of enzyme activity.
A. Competitive inhibition

- This type of inhibition occurs when the inhibitor binds

reversibly to the same site that the substrate would normally
occupy and, therefore, competes with the substrate for that
site. (Figure 11)

 Effect on Vmax

The effect of a competitive inhibitor is reversed by increasing

[S]. At a sufficiently high substrate concentration, the reaction
velocity reaches the Vmax observed in the absence of inhibitor
 Effect on Km

A competitive inhibitor increases the apparent Km for a given

Figure 7. Effects on Substrate concentration of reactions
substrate. This means that, in the presence of a competitive
velocity for an enzyme catalyzed reaction
inhibitor, more substrate is needed to achieve 1⁄2Vmax.

45
  Effect on the Lineweaver-Burk plot
- Competitive inhibition shows a characteristic Lineweaver-
Burk plot in which the plots of the inhibited and uninhibited
reactions intersect on the y-axis at 1/Vmax (Vmax is
unchanged).
- The inhibited and uninhibited reactions show different x-axis
intercepts, indicating that the apparent Km is increased in the
presence of the competitive inhibitor because -1/Km moves
closer to zero from a negative value
 Statin drugs as examples of competitive inhibitors
- This group of antihyperlipidemic agents competitively inhibits
the first committed step in cholesterol synthesis.
- This reaction is catalyzed by hydroxymethylglutaryl– CoA
reductase (HMG-CoA reductase).
- Statin drugs, such as atorvastatin (Lipitor) and pravastatin
(Pravachol), are structural analogs of the natural substrate for
this enzyme and compete effectively to inhibit HMG-CoA
reductase. (Figure 9)
By doing so, they inhibit de novo cholesterol synthesis, thereby
lowering plasma cholesterol level.
Figure 9. Statin drugs
B. Noncompetitive inhibition
- Noncompetitive inhibition occurs when the inhibitor and
substrate bind at different sites on the enzyme.
- The noncompetitive inhibitor can bind either free enzyme or
the ES complex, thereby preventing the reaction from
occurring. (Figure 11)
 Effect on Vmax
-Noncompetitive inhibition cannot be overcome by increasing
the concentration of substrate.
- Thus, noncompetitive inhibitors decrease the apparent Vmax
of the reaction.
 Effect on Km:
46
- Noncompetitive inhibitors do not interfere with the binding of
substrate to enzyme.
- Thus, the enzyme shows the same Km in the presence or
absence of the noncompetitive inhibitor.
 Effect on Lineweaver-Burk plot:
- Noncompetitive inhibition is readily differentiated from
competitive inhibition by plotting 1/vo versus 1/[S] and noting
that the apparent Vmax decreases in the presence of a
noncompetitive inhibitor, whereas Km is unchanged
 Examples of noncompetitive inhibitors
- Some inhibitors act by forming covalent bonds with specific
groups of enzymes.
- For example, lead forms covalent bonds with the sulfhydryl
side chains of cysteine in proteins.
- The binding of the heavy metal shows noncompetitive
inhibition.
- Ferrochelatase, an enzyme that catalyzes the insertion of Fe2+
into protoporphyrin (a precursor of heme) is an example of an
enzyme sensitive to inhibition by lead. (Figure 10)
Figure 11. Non-competitive Inhibitors
C. Enzyme inhibitors as drugs
- At least half of the ten most commonly dispensed drugs in the
United States act as enzyme inhibitors.
- For example, the widely prescribed beta-lactam antibiotics,
such as penicillin and amoxicillin, act by inhibiting enzymes
involved in bacterial cell wall synthesis.
Drugs may also act by inhibiting extracellular reactions. - This is
illustrated by angiotensin-converting enzyme (ACE) inhibitors.
Figure 11. A. Effect of a competitive inhibitor on the reaction
velocity (vo) versus substrate ([S]) plot. B. Lineweaver-Burk plot
of competitive inhibition of an enzyme.

REGULATION OF ENZYME ACTIVITY

- The regulation of the reaction velocity of enzymes is essential

if an organism is to coordinate its numerous metabolic
processes.
- The rates of most enzymes are responsive to changes in
substrate concentration, because the intracellular level of
many substrates is in the range of the Km.
- Thus, an increase in substrate concentration prompts an Figure 12. Effects of negative or positive effectors on an
increase in reaction rate, which tends to return the allosteric enzyme.
concentration of substrate toward normal
A. Regulation of allosteric enzymes A. Vmax is altered. B. The substrate concentration
that gives half-maximal
- Allosteric enzymes are regulated by molecules called effectors velocity (K0.5) is altered
(also called modifiers) that bind non- covalently at a site other
than the active site.  Heterotropic effectors
- These enzymes are usually composed of multiple subunits, - The effector may be different from the substrate, in which
and the regulatory (allosteric) site that binds the effector may case the effect is said to be heterotrophic.
be located on a subunit that is not itself catalytic. - For example, consider the feedback inhibition shown in.
- The presence of an allosteric effector can alter the affinity of (Figure 13)
the enzyme for its substrate or modify the maximal catalytic - The enzyme that converts D to E has an allosteric site that
activity of the enzyme, or both. binds the endproduct, G.
- If the concentration of G increases (for example, because it is
 Homotropic effectors not used as rapidly as it is synthesized), the first irreversible
-When the substrate itself serves as an effector, the effect is step unique to the pathway is typically inhibited.
said to be homotropic. - Feedback inhibition provides the cell with appropriate
amounts of a product it needs by regulating the flow of
- Most often, an allosteric substrate functions as a positive substrate molecules through the pathway that synthesizes
effector. that product.
- In such a case, the presence of a substrate molecule at one
site on the enzyme enhances the catalytic properties of the
other substrate-binding sites that is, their binding sites exhibit
cooperatively.
- These enzymes show a sigmoidal curve when reaction velocity
(vo) is plotted against substrate
concentration [S], (Figure 12)
47

Figure 13. Feedback inhibition of a metabolic pathway.
B. Regulation of enzymes by covalent modification
- Many enzymes may be regulated by covalent modification,
most frequently by the addition or removal of phosphate
groups from specific serine, threonine, or tyrosine residues
of the enzyme.
- Protein phosphorylation is recognized as one of the primary
ways in which cellular processes are regulated.
 Phosphorylation and dephosphorylation:
- Phosphorylation reactions are catalyzed by a family of
enzymes called protein kinases that use adenosine
triphosphate (ATP) as a phosphate donor.
- Phosphate groups are cleaved from phosphorylated enzymes
by the action of phosphoprotein phosphatases (Figure 14).
Figure 14. Covalent modification by the addition and removal
of phosphate groups
 Response of enzyme to phosphorylation:
- Depending on the specific enzyme, the phosphorylated form
may be more or less active than the unphosphorylated
enzyme.
- For example, phosphorylation of glycogen phosphorylase (an
enzyme that degrades glycogen) increases activity,
-Whereas the addition of phosphate to glycogen synthase (an
enzyme that synthesizes glycogen) decreases activity
- The regulatory mechanisms described above modify the
activity of existing enzyme molecules
- The increase (induction) or decrease (repression) of enzyme
synthesis leads to an alteration in the total population of
active sites.
48
- Enzymes subject to regulation of synthesis are often those
that are needed at only one stage of development or under
selected physiologic conditions.
ENZYMES IN CLINICAL DIAGNOSIS
- Plasma enzymes can be classified into two major groups. First,
a relatively small group of enzymes are actively secreted into
the blood by
certain cell types.
Regulation of enzymes by covalent modification
- Many diseases that cause tissue damage result in an increased
release of intracellular enzymes into the plasma.
- The activities of many of these enzymes are routinely
determined for diagnostic purposes in diseases of the heart,
liver, skeletal muscle, and other tissues.
- The level of specific enzyme activity in the plasma frequently
correlates with the extent of tissue damage
A. Plasma enzymes as diagnostic tools
- Some enzymes show relatively high activity in only one or a
few tissues.
- The presence of increased levels of these enzymes in plasma
thus reflects damage to the corresponding tissue.
-The appearance of elevated levels of ALT in plasma signals
possible damage to hepatic tissue.
-Increases in plasma levels of enzymes with a wide tissue
distribution provide a less specific indication of the site of
cellular injury and limits their diagnostic value.
B. Isoenzymes and diseases of the heart
- Most isoenzymes (also called isozymes) are enzymes that
catalyze the same reaction.
- Iso-enzymes may contain different numbers of charged amino
acids and may, therefore, be separated from each other by
electrophoresis. (Figure 15).
- The pattern of isoenzymes found in the plasma may,
therefore, serve as a means of identifying the site of tissue
damage.
- Different organs frequently contain characteristic proportions
of different isoenzymes.
 Quaternary structure of isoenzymes:
- Many isoenzymes contain different subunits in various
C. Induction and repression of enzyme
synthesis
combinations.
- For example, creatine kinase (CK) occurs as three isoenzymes.
- Each isoenzyme is a dimer composed of two polypeptides
(called B and M subunits) associated in one of three
combinations: CK1 = BB, CK2 = MB, and CK3 = MM.
- Each CK isoenzyme shows a characteristic electrophoretic
mobility (Figure 15).
 Diagnosis of myocardial infarction
- Measurement of blood levels of proteins with cardiac
specificity is used in the diagnosis of myocardial infarction (MI)
because myocardial muscle is the only tissue that contains
more than 5% of the total CK activity as the CK2 (MB)
isoenzyme.
-Appearance of this hybrid isoenzyme in plasma is virtually
specific for infarction of the myocardium.
- Troponin T and troponin 1 are regulatory proteins
involved in myocardial contractility.
-They are released into the plasma in response to cardiac
damage.
-Cardiac troponin I (cTnI) is highly sensitive and specific for
damage to cardiac tissue.
Figure 15. Subunit structure, electrophoretic mobility, and
enzyme activity of creatine kinase (CK) isoenzymes
49
Summary
Enzymes are protein catalysts that increase the velocity of
a chemical reaction by lowering the energy of the
transition state. Enzyme molecules contain a special
pocket or cleft called the active site. The active site
contains amino acid side chains that participate in
substrate binding and catalysis. The active site binds the
substrate, forming an enzyme–substrate (ES) complex.
Binding is thought to cause a conformational change in the
enzyme (induced fit) that allows catalysis. ES is converted
to enzyme-product (EP), which subsequently dissociates to
enzyme and product. An enzyme allows a reaction to
proceed rapidly under conditions prevailing in the cell by
providing an alternate reaction pathway with a lower free
energy of activation. Most enzymes show Michaelis-
Menten kinetics, and a plot of the initial reaction velocity
(vo) against substrate concentration ([S]) has a hyperbolic
shape similar to the oxygen dissociation curve of
myoglobin. Any substance that can diminish the velocity of
such enzyme- catalyzed reactions is called an inhibitor. The
two most commonly encountered types of reversible
inhibition are competitive (which increases the apparent
Km) and noncompetitive (which decreases the apparent
Vmax). In contrast, the multisubunit allosteric enzymes
frequently show a sigmoidal curve similar in shape to the
oxygen dissociation curve of hemoglobin. They typically
catalyze the committed step (often the rate-limiting or
slowest step) of a pathway. Allosteric enzymes are
regulated by molecules called effectors (also modifiers)
that bind noncovalently at a site other than the active site.
Effectors can be either positive (accelerate the enzyme-
catalyzed reaction) or negative (slow down the reaction).
An allosteric effector can alter the affinity of the enzyme
for its substrate, or modify the maximal catalytic activity of
the enzyme, or both.
Questions
In cases of ethylene glycol poisoning and its characteristic
metabolic acidosis, treatment involves correction of the
acidosis, removal of any remaining
ethylene glycol, and administration of an inhibitor of
alcohol dehydrogenase (ADH, alcohol:NAD+ oxidoreductase),
the enzyme that oxidizes ethylene glycol to
the organic acids that cause the acidosis. Ethanol (grain
alcohol) frequently is the inhibitor given to treat ethylene
glycol poisoning; it works by competitively inhibiting ADH. As a
competitive inhibitor, ethanol:
 A. increases apparent Km without affecting Vmax.
B. decreases apparent Km without affecting Vmax.
C. increases apparent Vmax without affecting Km.
D. decreases apparent Vmax without affecting Km.
E. decreases both apparent Vmax and apparent Km

ADH requires NAD+ for catalytic activity. In the reaction

catalyzed by ADH, an alcohol is oxidized to an aldehyde as
NAD+ is reduced to NADH and dissociates
from the enzyme. The NAD+ is functioning as a (an):

A. apoenzyme.
B. coenzyme-cosubstrate.
C. coenzyme-prosthetic group.
D. cofactor.
E. heterotropic effecto

A 70-year-old man was admitted to the emergency

room with a 12-hour history of chest pain. Serum creatine
kinase (CK) activity was measured at admission
(day 1) and once daily (Figure 5.24). On day 2 after admission,
he experienced cardiac arrhythmia, which
was terminated by three cycles of electric cardioconversion,
the latter two at maximum energy. [Note:
Cardioconversion is performed by placing two paddles, 12 cm
in diameter, in firm contact with the chest
wall and applying a short electric voltage.] Normal cardiac
rhythm was reestablished. He had no recurrence
of arrhythmia over the next several days. His chest pain
subsided and he was released on day 10. Which one of the
following is most consistent with the data presented?

A. The patient had a myocardial infarction 48 to 64 hours prior

to admission.
B. The patient had a myocardial infarction on day 2.
C. The patient had angina prior to admission.
D. The patient had damage to his skeletal muscle on day 2.
E. The data do not permit any conclusion concerning
myocardial infarction prior to, or after, admission to the
hospital.

50
 Adamson University May 24, 2019
Amino Acid Oxidation and the Production of Urea
SY 2018 – 2019 Patricia Alexandria Mae O. Canoza
Ms. Jemimah E. Sanggo, R.Ch, M.Sc.
Biochemistry Alain M. Damatac

Outline of the Lecture Metabolic Fates of Amino Groups

I. Introduction
II. Metabolic Fates of Amino Groups • Nitrogen, N2, is abundant in the atmosphere but is
III. Transamination and Deamination too inert for use in most biochemical processes.
IV. Urea Cycle • Amino acids derived from dietary protein are the
V. Fates Of Carbon Skeleton source of most amino groups.
VI. Diseases • Glutamate and glutamine acts as a kind of general
collection point for amino groups. In the cytosol of
Introduction hepatocytes, amino groups from most amino acids
The use of amino acids as fuel varies greatly by organism are transferred to alpha-ketoglutarate to form
• About 90% of energy needs of carnivores can be glutamate, which enters mitochondria and gives up
met by amino acids immediately after a meal its amino group to form NH4+. Excess ammonia
• Microorganisms scavenge amino acids from their generated in most other tissues is converted to the
environment for fuel when needed amide nitrogen of glutamine, which passes to the
• Only a very small fraction of energy needs of liver, then into liver mitochondria
herbivores are met by amino acids
• Plants do not use amino acids as a fuel source, but
can degrade amino acids to form other metabolites
Metabolic Circumstances of Amino Acid Oxidation
• Leftover amino acids from normal protein turnover
• Dietary amino acids that exceed body’s protein
synthesis needs
• Proteins in the body can be broken down to supply
amino acids for energy when carbohydrates are
scarce (starvation, diabetes mellitus)

FIGURE 2. Overview of the catabolic pathways of ammonia and

amino groups in vertebrates.
Fates of Nitrogen in Organisms
• Plants conserve almost all the nitrogen
• Many aquatic vertebrates release ammonia to their
FIGURE 1. Overview of amino acid catabolism in mammals. environment
In figure 1, it shows that amino groups and the carbon – Passive diffusion from epithelial cells
skeleton take separate but interconnected pathways. – Active transport via gills
• Many terrestrial vertebrates and sharks excrete nitrogen in
the form of urea
– Urea is far less toxic that ammonia
51
– Urea has very high solubility
• Some animals such as birds and reptiles excrete nitrogen as
uric acid
– Uric acid is rather insoluble
– Excretion as paste allows the animals to conserve water
• Humans and great apes excrete both urea (from amino
acids) and uric acid (from purines)
Excretory Forms of Nitrogen
The carbon atoms of urea and uric acid are highly oxidized; the
organism discards carbon only after extracting most of its
available energy of oxidation.
Dietary protein is enzymatically degraded through the
digestive tract. Chewing the food and mixing with saliva starts
the process of digestion
FIGURE 3. Part of the human digestive (gastrointestinal) tract.
Gastrin: when food enters the stomach, the gastric
mucosa secretes the hormone Gastrin that in turn stimulates
secretion of HCl by the parietal cells of the gastric glands.
52
Pepsinogen secretion from the Chief cells is also stimulated by
Gastrin and it is converted from this inactive, "zymogen" form
to active Pepsin
Proteins are cleaved on the amino­terminal side of aromatic
amino residues Tyrosine, Phenylalanine and Tryptophan by
Pepsin. As the contents pass into the small intestine, the
pancreas secretes bicarbonate to neutralize the acid and allow
other protein degrading enzymes to function.
Secretin: produced in the upper portion of the small intestine
(duodenum) and inhibits gastric acid secretion & stomach
motility; stimulates the pancreas to release bicarbonate ions
and stimulates the gall bladder to secrete bile.
The zymogen Trypsinogen is converted to the active protease
called Trypsin by an enzyme called enteropeptidase. Trypsin
then cleaves proteins at sites of Lysine and Arginine on the
carboxy­terminal side.
Chymotrypsinogen is converted to Chymotrypsin by Trypsin.
The Chymotrypsin then cleaves on the carboxy­terminal side
of Tyrosine, Phenylalanine and
Tryptophan. Elastase is formed from Proelastase by the action
of Trypsin and then cleaves proteins at bonds in which the
carboxyl group is contributed by small side chain amino acids
(alanine, glycine & serine). Carboxypeptidases are "broad
spectrum" enzymes and make multiple hits on remaining small
peptides.
The pancreas further protects itself against self-digestion by
making a specific inhibitor, a protein called pancreatic
trypsin inhibitor, that effectively prevents premature
production of active proteolytic enzymes within the pancreatic
cells.
Aminopeptidase that hydrolyzes successive amino-terminal
residues from short peptides.
Transamination and Deamination
A. Reactions involving the Amino Group:
1. Transamination
a. Reaction. catalyzed by enzymes called aminotransferases or
transaminases
Typically, α-ketoglutarate accepts amino groups
• L-Glutamine acts as a temporary storage of nitrogen
• L-Glutamine can donate the amino group when needed for
amino acid biosynthesis
 2. Deamination
a. Oxidative. Glutamate is transported from the cytosol to the
mitochondria where it undergoes Oxidative Deamination
catalyzed by L­glutamate dehydrogenase and alpha
ketoglutarate and ammonia (NH4+) are produced.
•
Can use either NAD+ or NADP+ as electron
acceptor
•
Ammonia is processed into urea for excretion
•
Pathway for ammonia excretion
transdeamination = transamination +
oxidative deamination

b. Mechanism. Pyridoxal phosphate (PLP) is the co­factor for

this class of reactions called Transamination. PLP is related to
Vitamin B6

FIGURE 4. Enzyme-catalyzed transaminations.

b. Non­Oxidative Deamination. The enzymatic conversion of

•Intermediate, enzyme-bound carrier of amino groups
Serine to Pyruvate + NH3 is an example of this type of reaction.
•Aldehyde form can react reversibly with amino groups
This is a Dehydration reaction with no net change in oxidation
•Aminated form can react reversibly with carbonyl groups
but results in the release of NH3.
Pyridoxal phosphate is covalently linked to the enzyme
in the resting enzyme by an internal aldimine.
The linkage is made via a nucleophilic attack of the
amino group of an active-site lysine.

3. Deamidation: Direct removal of an Amide functional group.

An example would be the second half of the reaction below.

The Glutamate that is formed channels these amino groups

into biosynthetic pathways or into terminal pathways where
the nitrogenous wastes are eliminated as Ammonia or Urea.

• Ammonia is safely transported in the bloodstream

as glutamine
• Excess ammonia in tissues is added to glutamate to
form glutamine (by glutamine synthetase).

53
 • Excess glutamine is processed in intestines,
kidneys, and liver (by glutaminase) liberating NH4+ in
mitochondria.
FIGURE 5. Ammonia transport in the form of glutamine.
Glutamate can donate ammonia to pyruvate to make alanine
• Vigorously working muscles operate nearly anaerobically
and rely on glycolysis for energy
• Glycolysis yields pyruvate
– if not eliminated lactic acid will build up
• This pyruvate can be converted to alanine for transport
into liver
54
The Glucose-Alanine Cycle
Alanine serves as a carrier of ammonia and of the carbon
skeleton of pyruvate from skeletal muscle to liver.
Excess ammonia is incorporated into Glutamate and then
transferred to pyruvate by the action of the enzyme Alanine
aminotransferase to form Alanine. The Alanine, with no net
charge at pH near 7, readily passes into the blood where it is
transported to the liver. In a reversal of the reaction that took
place in the muscle, Alanine is converted back to pyruvate and
the ammonia is transferred back to glutamate where it is
metabolized in the mitochondria to eventually be released as
urea.
Urea Cycle
Ammonia is highly toxic and must be utilized or excreted
• Free ammonia released from glutamate is converted to
urea for excretion.
• Carbamoyl phosphate synthetase I captures free ammonia in
the mitochondrial matrix
- First step of the urea cycle
- Regulated
- The first nitrogen acquiring reaction of the urea cycle
- Nitrogen from carbamoyl phosphate enters
the urea cycle
 Relationship between urea cycle and citric acid cycle
Entry of Aspartate into the Urea Cycle
-This is the second nitrogen-acquiring reaction.
1. CO2 needed for urea synthesis is mostly produced
from citric acid cycle.
2. Ammonia used for carbamoyl phosphate synthesis
(in mitochondria) is derived from glutamic acid by
glutamate dehydrogenase enzyme
3. Fumarate produced by urea cycle can be converted
into oxalacetic acid by citric acid cycle
4. Aspartic acid used in urea cycle is formed from
oxalacetic by transamination with glutamic acid by
AST (GOT) enzyme
5. ATP needed for urea cycle is derived from citric acid
cycle

The Reactions in the Urea Cycle

Aspartate –arginosuccinate shunt links urea cycle and citric acid

cycle

55
The reactions involved in the Urea Cycle are distributed – S-adenosylmethionine (adoMet)
between the liver mitochondria & the cytosol. – Biotin
Steps of urea biosynthesis • Biotin transfers CO2
1. Biosynthesis of carbamoyl phosphate
- This step occurs in mitochondria and needs
CO2, ammonia and phosphate and energy
CO2 is a product of citric acid cycle
- Ammonia is derived from glutamate by
deamination
- The phosphate and energy are derived from
ATP
- It is catalyzed by carbamoyl phosphate
synthetase-I THF is a versatile cofactor
- It needs biotin as a coenzyme. It needs • Tetrahydrofolate is formed from folate
magnesium ions also – an essential vitamin (B9)
- Carbamoyl phosphate synthetase I is • THF can transfer 1-carbon in different oxidation states – CH3,
activated by N-acetylglutamate CH2OH, and CHO
- Formed by N-acetylglutamate synthase • Used in a wide variety of metabolic reactions
when glutamate and acetyl-CoA • Carbon generally comes from serine
concentrations are high and activated by • Forms interconverted on THF before use
arginine
2. Formation of citrulline
- This step occurs in mitochondria
- It is catalyzed by ornithine
transcarbamoylase
3. Formation of argininosuccinate
- This step occurs in cytoplasm
- It is catalyzed by agininosuccinate
adoMet is better at transferring CH3
synthetase
• S-adenosylmethionine is the preferred cofactor for methyl
- It utilizes one ATP and 2 high energy bonds
transfer in biological reactions
4. Cleavage of argininosuccinate
– Methyl is 1000 times more reactive than THF methyl group
- This step occurs in cytoplasm
• Synthesized from ATP and methionine
- It is catalyzed by argininosuccinase enzyme
- Argininosuccinate is cleaved into arginine
and fumarate
- Fumarate produced is used to regenerate
aspartic acid again
5. Cleavage of arginine
- This step occurs in cytoplasm
- It is catalyzed by arginase enzyme
- Arginine is cleaved to urea and ornithine

In one turn of urea cycle:

• 2 molecules of ammonia are consumed

• 1 molecule of carbon dioxide is consumed
• 1 molecule of urea is formed
Fates of Carbon Skeleton
• A molecule of ornithine is regenerated for another turn of
the cycle. Amino acids can be classified according to the metabolic fate of
• 3 ATP are consumed. the carbon skeleton in:
• 4 high-energy bonds are consumed. – ketogenic,
– glucogenic
Several cofactors are involved in amino acid catabolism – ketogenic and glucogenic

• Important in one-carbon transfer reactions Ketogenics: Amino acids that yield acetyl CoA or acetoacetyl
– Tetrahydrafolate (THF) CoA ( e.g. they do not produce metabolites that can be

56
converted in glucose). Lysine and Leucine are the only amino
acids that are exclusively ketogenics.
Glucogenic: Amino acids whose catabolism yields to the
formation of Pyruvate or Krebs Cycle metabolites, that can be
converted in glucose through gluconeogenesis.
9. Amino acids serve as precursors for very important
biological amines.
10. Some amino acids are ketogenic, some are glucogenic and
some are both. Ketogenic amino acids are degraded to
acetoacetyl­CoA and/or acetyl­CoA that can be converted to
ketone bodies.

Glucogenic amino acids are: Alanine, Arginine, Asparagine, QUESTIONS:

Aspartate, Cysteine, Glutamate, Glycine, Histidine, 1. Which of the following amino acids is considered as both
Methionine, Proline, Serine, and Valine ketogenic and glucogenic?
A. Valine
Glucogenic and ketogenic: Amino acids that yield some B. Tryptophan
products that can become glucose and others that yields C. Lysine
acetyl CoA or Acetoacetyl CoA. D. None of these

Amino acids of this kind are Isoleucine, Phenylalanine, 2. A person with phenylketonuria cannot convert
Tryptophan, Tyrosine and Threonine. A. phenylalanine to tyrosine
B. phenylalanine to isoleucine
C. phenol into ketones
Diseases
D. phenylalanine to lysine
Ammonia Intoxication
• Ammonia is highly toxic to the central nervous 3. In deamination, amino acid is converted into
system Blood ammonia level 10 – 60 ug/dl A. aldol acid
• Accumulation of ammonia in the body leads to B. keto acid
nervous manifestations and may be fatal. C. hydrochloric acid
D. carboxylic acid
Causes of hyperammonemia 4. Process of breakdown of amino acids to α keto acids is
1. Acquired hyperammonemia called
• Liver cell failure A. cisamination
• Shunt operations between portal and systemic circulation B. amination
2. Congenital hyperammonemia C. transamination
Defect in any one of the five enzymes of urea cycle leads to D. Racemization
ammonia intoxication. e.g.
• Hyperammonemia type-I due to defect in 5. A ketogenic amino acid is one which degrades to
carbamoyl phosphate synthetase-I
• Hyperammonemia type-II due to defect in ornithine A. keto-sugars
transcarbamoylase enzyme B. either acetyl CoA or acetoacetyl CoA
C. pyruvate or citric acid cycle intermediates
Summary D. multiple intermediates including pyruvate or citric acid
1. Dietary proteins are the primary source of biologically cycle intermediates and acetyl CoA or acetoacetyl CoA
useful nitrogen in our bodies.
2. The general scheme for the further metabolism of
"digested" amino acids involves the transfer of the amino
group to alpha­ketoglutarate forming glutamate plus an
alpha­keto acid.
3. The glutamate produced is transported to liver
mitochondria and deaminated by glutamate dehydrogenase.
4. Glutamine and alanine transport ammonia formed in other
tissues to the liver.
5. Nitrogen is excreted as ammonia or urea. High serum levels
of ammonia could indicate liver disease.
6. Urea is formed from ammonia in a series of reactions called
the urea cycle.
7. Deaminated amino acids produce carbon skeletons that can
be funneled into the citric acid cycle.
8. Amino acids can serve as important sources of energy.

57
 Adamson University May 24, 2019
SY 2018 – 2019 Nucleic Acid A1 De Asis, Zsar Alanee D.
Biochemistry Ms. Jemimah E.Sanggo, R.Ch, M.Sc. Racho, Marnie Martha

Outline of the Lecture sugar covalently linked together

I. Levels of Structure in Nucleic Acid - differs from a nucleotide by lacking a
II. Structure of DNA phosphate group in its structure
III. Other Conformations of Double Helix
IV. Supercoiling - a base forms a glycosidic linkage with the
V. Denaturation of DNA linkage with the sugar
VI. Kinds of RNA
VII. RNA Structure
VIII. Summary β-D-ribose– resulting compoundis aribonucleoside
IX. Disease β-D-deoxyribose– resulting compound is adeoxyri-
X. Question ribonucleoside

Levels of Structure in Nucleic Acid

primary structure of nucleic acids - order of bases in the

polynucleotide sequence
secondary structure - three-dimensional conformation of the
backbone
tertiary structure - the supercoiling of the molecule

Two Principal Types of Nucleic Acids

1. DNA (deoxyribonucleic acid)
2. RNA (ribonucleic acid)
The Covalent Structure of Polynucleotides
Comparison of the structures of a ribonucleoside and a
Nucleotides - monomers of nucleic acid
deoxyribonucleoside
• consist of three parts—a nitrogenous base,
a sugar and a phosphoric acid residue
(covalently bonded together) Note: The glycosidic linkage is from the C-1' carbon
Nucleic Acid Bases -also called nucleobases of the sugar to the N-1 nitrogen of pyrimidines or
• refers to a one-ring or two-ring to the N-9 nitrogen of purines.
nitrogenous aromatic compounds
Note: The ring atoms of the base and the carbon
Two Types of Nucleic Acid Bases
1. Pyrimidines– single-ring aromatic compounds atoms of the sugar are both numbered and the
2. Purines – double-ring aromatic compounds numbers of the sugar atoms are primed to
prevent confusion.

Pyrimidine Bases
• when phosphoric acid is esterified to one of the
hydroxyl groups of the sugar portion of a nucleoside, a
nucleotide is formed

Purine Bases

nucleoside - a compound that consists of a base and a

58
 • Each nucleotide has a phosphate group in its structure

5' nucleotides - most commonly encountered in nature

polymerization of nucleotides - gives rise to nucleic acids

Note: When nucleotides are joined by

phosphodiester bonds, they form a sugar –
phosphate backbone, giving rise to DNA and RNA.

THE STRUCTURE OF DNA

DNA - consists of two polynucleotide chains wrapped

around each other to form a helix

Hydrogen bonds - determine the alignment of the helix, with

the paired bases lying in planes perpendicular to the helix axis

Sugar-phosphate backbone - outer part of the helix

The double helix

Note: The amount of A was always the same as

the amount of T, and that the amount of G
always equaled the amount of C.

The structures and names of the commonly occurring Note: The chains run in anti-parallel directions,
nucleotides
one 3' to 5' and the other 5' to 3.'
59
X-ray diffraction pattern of DNA - demonstrated the helical
structure and the
diameter

Comparison of the A, B, and Z forms of DNA

• Since complementary base pairing occurs along the entire
double helix, the two chains are also referred to as
A-DNA
complementary strands.
• right-handed helix
• An adenine–thymine (A–T) base pair has two hydrogen
• base pairs have a marked propeller-twist with respect to
bonds between the bases; a guanine–cytosine (G–C) base
the helix axis
pair has three hydrogen bonds
• originally found in dehydrated DNA samples
B-DNA
• right-handed helix
• the base pairs lie in a plane that is close to perpendicular
to the helix axis
• the normal, physiological DNA form
Z-DNA
• left-handed helix
• considered a derivative of the B form of DNA
• often occurs when there is a sequence of alternating
purine - pyrimidine, such as dCpGpCpGpCpG

Supercoiling

Base Pairing

• The inside diameter of the sugar–phosphate backbone of

the double helix is about 11 Å (1.1 nm)
• The distance between the points of attachment of the
bases to the two strands of the sugar–phosphate
backbone is the same for the two base pairs (A–T and G–
Supercoiled DNA Topology
C), about 11 Å (1.1 nm)
• The outside diameter of the helix is 20 Å (2 nm)
• Tertiary structure of DNA depends on supercoiling.
• The length of one complete turn of the helix along its
Circular DNA is twisted before the circle is sealed which
axis is 34 Å (3.4 nm) and contains 10 base pairs
gives rise to supercoiling, in prokaryotes. In eukaryotes,
the supercoiled DNA is complexedwith proteins known
Other Conformation of Double Helix
as histones

60
 Denaturation of DNA • AminoacyltRNA – amino acid brought to the assembly site
covalently bonded to tRNA
• Translation – process which the order bases in mRNA
specifies the order of amino acids in the growing protein.
; genetic message

DNA Denaturation – a process of beaking double stranded

DNA into single strand

Hyperchromicity – As strands are separate, the wavelength of

absorption does not change.
• Monitored by observing the absorption of ultraviolet
light
• Bases absorbs light in the 260nm wavelength region
• When DNA is replicated, it becomes single stranded and
complementary bases can be aligned
The Fundamental Process fof Information Transfer In Cells
Melting Temperature (Tm) – the heat of denaturation of DNA
• G–C base pair has 3 hydrogen bonds (more In Prokaryotes,
hydrophobic) and A–T base pair has 2.
Transcription
• Tm depends on its base composition; higher Tm is rich in mRNA protein
G–C base pairs.

Kinds of RNA
The base sequences of all types of RNA are determined by that
of DNA.

Role of mRNA in Transcription

In Eukayotes,

Different kind of RNA

• Transcription – process which the order of bases passed

from DNA to RNA
• Ribosomes – rRNA is associated w/ proteins; sites for
assembly of the growing polypeptide chain in protein
synthesis.
Role of mRNA in Transcription
61

• Introns – spicing out intervening sequences
• Exons – parts of mRNA that will be experessed
• Small Nuclear RNA found only in eukaryotic cell
RNA Structure
Transfer RNA (tRNA) – smallest RNA
• single-stranded polynucleotide chain, between 73
and 94 nucleotide residues long, and has a
molecular mass of about 25,000 Da
• occurs the intrachain hydrogen bonding
• the formation of A–U and G–C base pairs similar in
DNA
• The duplexes formed have a A-helical form
• It has a clover leaf structure
 Stems - hydrogen-bonded portions of the
molecule
 Loops - non-hydrogen-bonded portions
In protein stynthesis, both tRNA and mRNA bound to the
ribosome in a definite spatial arrangement that ultimately
ensures the correct order of the amino acids in the growing
polypeptide chain.
Ribosomal RNA (rRNA) – molecules tend to be quite large
• RNA portion of a ribosome is 60%–65% of the total
weight, and the protein portion is 35%–40% of the
weight.
• In both prokaryotes and eukaryotes, ribosome
consists of two subunits:
 Smaller subunits consists of one large RNA
molecule and about 20 different proteins
62
 Larger subunit consists of two RNA molecules
in prokaryotes (three in eukaryotes) and
about 35 different proteins in prokaryotes
(about 50 in eukaryotes)
Structure of typical prokaryotes ribosome
Summary
• The order of bases is the primary structure of nucleic
acids, the secondary structure is the three-
dimensional conformation of the backbone and the
tertiary structure is the supercoiling of the molecule
• two kinds of nitrogen-containing nucleobases,
pyrimidines and purines, are joined to sugars to form
nucleosides
• Ehe two strands of the double helix can be separated
by heating DNA samples. This process is called
denaturation.
• DNA denaturation can be monitored by observing the
rise in ultraviolet absorption than accompanies the
process.
• The temperature at which DNA becomes denatured
by heat depends on its base composition; higher
 temperatures are needed to denature DNA rich in G– Questions
C base pairs. 1. What are the components and structures of
• Four kinds of RNA—transfer RNA, ribosomal RNA, nucletides?
messenger RNA, andsmall nuclear RNA—are involved 2. What is the nature of the DNA double helix?
in protein synthesis. 3. What is the difference of prokaryotes and eukayotes
• Transfer RNA transports amino acids to the sites of sequence in RNA?
protein synthesis on ribosomes, which consist of 4. What are the symptoms of Lesch-Nyhan Syndrome ?
ribosomal RNAs and proteins. 5. What is hyperchromicity ?
• Messenger RNA directs the amino acid sequence of
proteins.
• Smallnuclear RNA is used to help process eukaryotic
mRNA to its final form.
• RNA interference, which requires short stretches of
siRNA, controls gene expression.

Disease

Lesch-Nyhan syndrome is a rare inborn error of purine

metabolism which is characterized by the deficiency of the
activity of the enzyme hypoxanthine-guanine
phosphoribosyltransferase (HPRT). When HPRT is not present,
the purines hypoxanthine and guanine are not built into
nucleotides. Uric acid levels are abnormally high and sodium
urate crystals may abnormally accumulate in the joints and
kidneys. This syndrome is inherited as an X-linked recessive
genetic disorder which oftenaffects males.

The symptoms of Lesch-Nyhan syndrome include impaired

kidney function, acute gouty arthritis, and self-mutilating
behaviors such as lip and finger biting and/or head banging.
Additional symptoms include involuntary muscle movements,
and neurological impairment.

63
 Adamson University May 24, 2019
SY 2018 – 2019 Nucleic Acid Metabolism Joshua Rhoger M. Abando
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Eduel Gerard D. Bucio
Outline of the Lecture
I. Nucleic Acid Metabolism
II. Nucleosides and Nucleotides
III. Nitrogen Bases i.e. purines and pyrimidines
IV. Synthesis of Nucleic Acids
V. Degradation of Nucleic Acids
VI. Nucleic Acid Disorders

Nucleic Acid Metabolism

– It is a process by which nucleic acids (DNA and RNA) are
synthesized and degraded.
Nucleic Acid
– Polymer of nucleotides: highly
flexible compared to peptides.
– Universal in living things, as Table 2: Classifications of Nucleotide
Nucleoside Phosphoric Acid Nucleotide
they are found in all cells
Adenosine Phosphoric Acid Adenylate (AMP)
– It were first discovered by
Guanosine Phosphoric Acid Guanylate (GMP)
Friedrich Miescher in 1871.
Thymidine Phosphoric Acid Thymidylate (TMP)
Cytidine Phosphoric Acid Cytidylate (CMP)
Nucleosides and Nucleotides
Nucleoside Uridine Phosphoric Acid Uridylate (UMP)
– Glycosylamines that can be brought of as nucleotides
without a phosphate group. Nitrogen Bases
– A structure formed by the combination of nitrogen base Purines Pyrimidines
and five-carbon sugar.

– They both contain a sugar and a phosphate, but have

nitrogenous bases that are different sizes.
– In more complex multicellular animals, they are both
Composition of nucleotides, which make up nucleic acids
primarily produced in the liver.

IMPORTANT PURINES AND PYRIMIDINES

Table 1: Classifications of Nucleoside
N2 base Sugars Nucleoside – Adenine and Guanine are the principal purines of both
Adenine Deoxyribose/Ribose Adenosine DNA and RNA
Guanine Deoxyribose/Ribose Guanosine – Cytosine and Thymine are the pyrimidines that occur in
Thymine Deoxyribose Thymidine DNA while Cytosine and Uracil are the pyrimidines in RNA.
Cytosine Deoxyribose/Ribose Cytidine – Caffeine (coffee) and Theobromine (coffee and tea) are
Uracil Ribose Uridine naturally occurring purines.
* Suffix: “osine” for purine bases
* Prefix: “deoxy” for DNA

Nucleotide
– Building blocks of Nucleic Acids (DNA and RNA).
– It is phosphoric esters of nucleosides.
Adenine Guanine Cytosine Thymine Uracil
– A structure formed by the combination of nucleobase, a
five-carbon sugar and one or more phosphate groups.

64
 Synthesis of Nucleic Acids Pyrimidine Synthesis (De novo synthesis)
Purine Synthesis (De novo synthesis)

Location: De novo pyrimidine synthesis occurs in the cytosol of

cells in all tissues.
 Substrates: CO2; glutamine; ATP; Aspartate; H2O;
NAD+; Phosphoribosyl pyrophosphate (PRPP).
 Products: UTP; CTP; glutamate; NADH; CO2

Important Enzymes and Regulation

• Carbamoyl phosphate synthetase II: Inhibited by
UTP; activated by ATP and PRPP.
• Orotidylate (OMP) decarboxylase: Inhibited by
 PRPP synthetase is the rate limiting step in the
UMP and CMP.
synthesis of purines.
• TP synthetase: Inhibited by CTP.
 Glutamine: PRPP amidotransferase catalyzes the first-
committed step in purine synthesis.
Degradation of Nucleic Acids
 IMP branch to AMP Salvage Pathway for Purines
o Inhibitor: AMP
o Need for GTP
 IMP branch to GMP
Inhibitor: GMP
Need for ATP

Location: Purine synthesis occurs in all tissues. The major site of

purine synthesis is in the liver and, to a limited extent, in the
brain.
 Substrates: Ribose-5-phosphate; glycine; glutamine;
H2O; ATP; CO2; aspartate.
 Products: GMP; AMP; glutamate; fumarate; H2O.

Important Enzyme and Regulation

• PRPP synthetase: inhibited by AMP, IMP, GMP
• Glutamine PRPP amidinotransferase: Inhibited by
AMP, IMP, and GMP.
• IMP dehydrogenase: Inhibited by GMP.
• Adenylosuccinate synthetase: Inhibited by AMP.

65
  What: Recycling of products of nucleic acid Purine Disorders
breakdown.  Hyperuricemia – excess uric acid in the blood.
 Where: Occurs primarily in extraphetic tissues.  Hypouricemia – level of uric acid in blood serum that
 How: From free bases (A,G). is below normal.
 Who: APRT and HGPRT is responsible for most of the  Gout – form of arthritis caused by excess uric acid in
recycling. the bloodstream.
 How much: Accounts for 90% daily purine synthesis.  Lesch-Nyhan Syndrome – a rare inherited disorder
 Why: Most purine bases are recycled rather than caused by deficiency of the enzyme (HGPRT).
degraded.  Von Gierke disease – a condition in which the body
cannot break down glycogen.
Location: Purine synthesis via the salvage pathways occurs in all  Adenosine deaminase deficiency – an autosomal
tissues. It is especially important in the brain and the bone recessive metabolic disorder that causes
marrow. immunodeficiency.
• Substrates: Hypoxanthine; PRPP; guanine; adenine.  Purine nucleoside deficiency – have low numbers of
• Products: GMP; AMP; IMP. immune system cells called T-cells.

Important enzymes and Regulation Pyrimidine Disorders

• HGPRT: Inhibited by IMP and GMP.  Orotic Acidurias – decreased in ability to synthesize
• Adenine phosphoribosyltransferase: Inhibited by AMP. pyrimidines. The only known enzyme deficiency of the
de novo pyrimidine synthesis.
Salvage Pathway for Pyrimidines  Reye’s syndrome – secondary orotic aciduria. Diverted
for the increased synthesis and excretion of orotic
acid.

Summary
 Nucleic Acid Metabolism is a process by which nucleic
acids (DNA or RNA) are synthesized and degraded.
 The monomeric unit of nucleic acid consists of
nucleoside (without phosphate group) and nucleotide
(with phosphate group).
 Nucleotides can be separated into two: Purines and
Pyrimidines that contain a sugar and phosphate group
but differ in nitrogenous bases.
 Nucleotide synthesis is an anabolic mechanism while
nucleotide degradation is a catabolic reaction.
 Different nucleic acid disorders were introduced as an
effect of deficiency in enzymes.

Questions
1. It is a structure formed by the combination of nucleobase,
five-sugar unit and a phosphate group called?
2. What are the pyrimidines that occur in RNA?
3. Where does major site for synthesis of purines occur?
4. TP synthetase is inhibited by what enzyme?
5. It is a a rare inherited disorder caused by deficiency of the
 Only 30% of pyrimidine nucleotides are provided by enzyme (HGPRT) called?
the salvage pathway.
 Pyrimidine nucleosides that travel in blood are taken END.
up by tissues.

Location: Pyrimidines can be salvaged from orotic acid, uracil,

and thymine but not from cytosine. Salvage is accomplished by
the enzyme pyrimidine phosphoribosyl transferase.
Nucleic Acid Disorders

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 Adamson University
SY 2018 – 2019 Photosynthesis: Dark Reaction May 24, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Accad, NMD., Garcia, MR
Outline of the Lecture 4Xu5P 4Ru5P: Phosphopentose epimerase
I. Dark Reactions of Photosynthesis 2R5P 2Ru5P: Phosphopentose isomerase
II. Calvin Cyle
III. CO2 Fixation in Tropical plants
D. Phosphorylation:
6Ru5P + 6ATP 6RBP + 6ADP: Phosphoribulose
Dark Reactions of Photosynthesis
kinase
– Polysaccharides : The actual storage form carbon dioxide
by photosynthesis
– Stroma: the location in the plant’s cell where CO2 fixation
takes place
– Reactions pathways are similar to glycolysis and pentose
pathway but only uses ATP and NADPH.
– Light independent instead, uses ATP for energy and
NADPH provides the electrons required to fix CO2.
– Needs 6 molecules of CO2 to produce to produce one
molecule of glucose (refer to the equation)
– The overall reaction pathway for dark reaction is cyclic
and is called the Calvin cycle.

Equation for the overall reaction:

6CO2 + 12NADPH + 18ATP Glucose + 12NADP++ 18ADP
+18Pi

CO2 Fixation in Tropical Plants

– Tropical plants has a C4 pathway, also known as Hatch-
Slack pathway, that leads to the C3 pathway of the Calvin

Figure 2. Calvin Cycle diagram

Cycle.
– Corn is an example of C4 plant.
– C4 pathway fixes CO2 in the mesophyll cells to unfix in the
bundle sheath where CO2 enters the C3 pathway
– Oxaloacetate is reduced to malate
– Photorespiration: Oxygen is used instead of CO2 during
Figure 1. Chloroplast structure showing the stroma the reaction catalyzed by rubisco.
– Photorespiration happens in C3 plants
Calvin Cycle – CAM (Crassulacean Acid Metabolism) photosynthesis
– Also known as Benson-Calvin cycle / light independent happens in plants in dry conditions (e.g. cactus)
reactions
– takes place after light has been captured by sunlight Q: 1. Other name for calvin cycle
– the main purpose of the Calvin cycle: produce glucose 2. Main purpose of calvin cycle
from CO2. 3. how many turns of calvin cycle is needed to produce
– Divided into three main stages: glucose
4. example of C3 plants
A. Preparation:
5. example of C4 plants
5G3P 5DHAP: Triose phosphate isomerase
3G3P + 3DHAP 3FBP: Aldolase
3FBP + 3H2O +3F6P 1 3Pi Benson Calvin, produce glucose, six, (rice, wheat), (sugarcane, corn)

B. Reshuffling:
2F6P + 3G3P 2Xu5P + 2R5P : Transketolase
2E4P + 2DHAP 2SBP: Aldolase

C. Isomerization:
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 Adamson University
SY 2018 – 2019 PHOTOSYNTHESIS May 23, 2019
Biochemistry Ms. Jemimah E. Sanggo, R.Ch, M.Sc. Garcia, Mitzi Ramiah S.
Outline of the Lecture
I. Photosynthesis: Harvesting Light Energy – Embedded in the thylakoid membranes (lamellae) are
II. General Features of Photophosphorylation photosynthetic pigments and enzyme complexes that
III. Light Absorption
IV. The Central Photochemical Event: Light- Driven carry out the light reactions and ATP synthesis.
Electron Flow – The stroma (the aqueous phase enclosed by inner
V. ATP Synthesis by Photophosphorylation membrane) contains most of the enzyme required
VI. The Evolution of Oxygenic Photosynthesis
VII. Summary for the dark reactions.

Photosynthesis: Harvesting Light Energy

Capture of solar energy by photosynthetic organisms and its

conversion to the chemical energy of reduced organic
compounds is the ultimate source of nearly all biological
energy.

– Photosynthetic organisms trap solar energy and form

ATP and NADPH, which they use as energy sources to
make carbohydrates and other organic compounds
from CO2 and H2O; simultaneously, they release O2
into the atmosphere.
Schematic diagram of Chloroplast.
– Photosynthesis occurs in a variety of bacteria and in
unicellular eukaryotes (algae) as well as in vascular
– Robert Hill: when leaf extracts containing
plants.
chloroplasts were illuminated, they (1) evolvedO2
– Overall equation for photosynthesis in vascular plants
and (2) reduced a nonbiological electron acceptor
describes an oxidation-reduction.
added to the medium.
CO2 + H2O O2 + (CH2O)
Hill reaction:
2H2O + 2A 2AH2 + O2
General Features of Photophosphorylation
A is the artificial electron acceptor, or Hill reagent
– H2O poor electron donor
– Proved that (1) absorbed light energy causes
– Photophosphorylation differs from oxidative
electrons to flow from H2O to an electron acceptor
phosphorylation in requiring the input of energy in
and (2) CO2 was neither required nor reduced to a
the form of light to create a good electron donor and
stable form under these conditions; thus, O2
electron acceptor.
evolution could be dissociated from CO2 reduction.
Photosynthesis in plants encompasses two processes:
– Severo Ochoa: NADP+ is the biological electron
– Light-dependent reactions: occur when plants are
acceptor in chloroplasts.
illuminated.
2H2O + 2NADP+ 2NADPH + 2H+ + O2
 Chlorophyll and other pigments of photosynthetic
cells absorb light energy, conserve it as ATP and
Light Absorption
NADPH, O2 simultaneously evolved.
– Carbon-assimilation reactions: Carbon-fixation
Chlorophyll
reactions or dark reactions
 ATP and NADPH are used to reduce CO2 to form
– Most important light-absorbing pigments in the
triose phosphates, starch, and sucrose, and other
thylakoid membranes
products.
– Green pigments with polycyclic, planar structures
– In photosynthetic eukaryotic cells both processes
resembling protoporphyrin of hemoglobin but with
happen in chloroplast.
Mg2+, not Fe2+, in central position.
– has an extended polyene structure, with alternating
single and double bonds, which characteristically
Light Drives Electron Flow in Chloroplasts
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 show strong absorption in the visible region of the
spectrum.
– Have unusually high molar extinction coefficients
therefore are well-suited for absorbing visible light
during photosynthesis.
– Contain chlorophyll a and chlorophyll b: absorption
spectra are different that they complement each
other’s range of light absorption

Light-harvesting complexes (LHCs): specific binding

proteins, which chlorophyll molecules are fixed in
relation to each other, other protein complexes,
and to the membrane.
Example: Phycobilins for cyanobacteria and red algae

Accessory pigments (Carotenoids)

– In thylakoid membranes.
– Absorb light at wavelengths not absorbed by the
chlorophylls thus, supplementary light receptors.
– most important are 𝛽𝛽-carotene (red-orange
isoprenoid), and lutein (yellow).

Chlorophyll Funnels the Absorbed Energy to Reaction Centers

by Exciton Transfer

– Exciton: quantum of energy passed from an excited

molecule to another molecule in a process called
exciton transfer.
– Photosystems

 light-absorbing pigments of thylakoid or bacterial

membranes arranged in functional arrays.
 Its pigments are called light-harvesting or antenna
molecules
 All pigment molecules can absorb photons, but only
chlorophyll molecules associated with the;
 Photochemical reaction center are specialized to
transduce light into chemical energy.

Exciton and electron transfer

69

Exciton and electron transfer (continued)
– Generalized scheme shows conversion of the energy
of an absorbed photon into separation of charges at
the reaction center. Note that step 1 may repeat
between successive antenna molecules until the
exciton reaches a reaction-center chlorophyll. The
asterisk (*) represents the excited state of an
antenna molecule.
excitation by light causes electric charge separation and
initiates an oxidation-reduction chain.
The Central Photochemical Event: Light- Driven Electron
Flow
Bacteria Have One of Two Types of Single Photochemical
Reaction Center
– Photosynthetic bacteria have simple
phototransduction machinery, with one of two
general types of reaction center: The Pheophytin-
Quinone Reaction Center and the Fe-S Reaction
Center.
The Pheophytin-Quinone Reaction Center (Type II Reaction
Center)
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– Structural studies of the reaction center of a purple
bacterium provided information about light-driven
electron flow from an excited special pair of
chlorophyll molecules, through pheophytin, to
quinones.
– light energy drives electrons from the reaction-center
P870 through pheophytin (Pheo), a quinone (Q), and
the cytochrome bc1 complex, then through
cytochrome c2 and back to the reaction center.
Electron flow through the cytochrome bc1 complex
causes proton pumping, creating an electrochemical
potential that powers ATP synthesis.
Functional modules of photosynthetic machinery in
purple bacteria.
The Fe-S Reaction Center (Type I Reaction Center)
– An alternative path, in green sulfur bacteria, sends
electrons from reduced quinones to NAD+
– have two routes for electrons driven by excitation of
P840:
 a cyclic route through a quinone to the cytochrome
bc1 complex and back to the reaction center via
cytochrome c.
  a noncyclic route from the reaction center through
the iron-sulfur protein ferredoxin (Fd), then to
NAD+ in a reaction catalyzed by ferredoxin: NAD
reductase.
Functional modules of photosynthetic machinery in green
sulfur bacteria.
In Plants, Two Reaction Centers Act in Tandem
– Photosynthetic apparatus of modern cyanobacteria,
algae, and vascular plants is more complex than the
one-center bacterial systems. They have two
systems:
 Photosystem II (PSII) is a pheophytin-quinone type
of system.
 Photosystem I (PSI) is structurally and functionally
related to the type I reaction center of green sulfur
bacteria.
– Flow of electrons through the photosystems
produces NADPH and ATP, in the ratio of about 2:3.
– Second type of electron flow produces ATP only and
allows variability in the proportions of NADPH and
ATP formed.
– Localization of PSI and PSII between the granal and
stromal lamellae can change and is indirectly
controlled by light intensity, optimizing the
71
distribution of excitons between PSI and PSII for
efficient energy capture.
Water Is Split by the Oxygen-Evolving Complex
– The light-driven splitting of H2O is catalyzed by a Mn-
containing protein complex; O2 is produced.
– The reduced plastoquinone carries electrons to the
cytochrome b6f complex; from here they pass to
plastocyanin, and then to P700 to replace those lost
during its photoexcitation.
– Electron flow through the cytochrome b6f complex
drives protons across the plasma membrane, creating
a proton-motive force that provides the energy for
ATP synthesis by an ATP synthase.
ATP Synthesis by Photophosphorylation
Process by which the proton gradient by the two plant
photosystems drives the synthesis of ATP, the other energy
conserving product of the light-dependent reactions
A Proton Gradient Couples Electron Flow and
Phosphorylation
1. The reaction centers, electron carriers, and ATP-forming
enzymes are located in a proton-impermeable membrane,
thylakoid membrane- must be intact to support
photophosphorylation.
2. Photophosphorylation can be uncoupled from electron
flow by reagents that promote the passage of protons through
the membrane.
3. Photophosphorylation can be blocked by venturicidin
and others that inhibit the formation of ATP from ADP and Pi
by the mitochondrial ATP synthase
4. ATP synthesis is catalyzed by FoF1 complexes, located on the
outer surface of the thylakoid membranes.
Comparison of the topology of proton movement and ATP
synthase orientation in the membranes of mitochondria,
chloroplasts, and the bacterium E. coli.
 The Evolution of Oxygenic Photosynthesis

– Chloroplasts evolved from ancient photosynthetic

bacteria
 Chloroplasts in modern organisms resemble
mitochondria in several properties, and are
believed to have originated by the same
mechanism that gave rise to mitochondria:
endosymbiosis.
– In Halobacterium, a single protein absorbs light and
pumps protons to drive ATP synthesis
– Many photosynthetic microorganisms obtain
electrons for photosynthesis not from water but from
donors such as H2S.
– An entirely different mechanism for converting light
energy to a proton gradient has evolved in the
modern archaea, in which the light-harvesting
pigment is retinal.
Summary

Photosynthesis and Photophosphorylation

– encompasses two processes: Light-dependent
reactions and Carbon-assimilation reactions involving
the Calvin cycle.
Light Absorption
– Photophosphorylation in the chloroplasts of green
plants and in cyanobacteria involves electron flow
through a series of membrane-bound carriers.
The Central Photochemical Event: Light- Driven Electron Flow
– Bacteria have a single reaction center; in purple
bacteria, it is of the pheophytin-quinone type, and in
green sulfur bacteria, the Fe-S type.
– Cyanobacteria and plants have two different
photoreaction centers, arranged in tandem,
Photosystem I and II
ATP Synthesis by Photophosphorylation
– In plants, both the water-splitting reaction and
electron flow through the cytochrome b6f complex
are accompanied by proton pumping across the
thylakoid membrane. The proton-motive force thus
created drives ATP synthesis by a CFoCF1 complex
similar to the mitochondrial FoF1 complex.
– The ATP synthase of chloroplasts (CFoCF1) is very
similar in both structure and catalytic mechanism to
the ATP synthases of mitochondria and bacteria.
Physical rotation driven by the proton gradient is
accompanied by ATP synthesis at sites that cycle
through three conformations, one with high affinity
for ATP, one with high affinity for ADP + Pi, and one
with low affinity for both nucleotides.

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