Biochemistry Review for the NBDE 2008 November 5, 2008

Janeen Arbuckle, PhD

Contact Information:

janeen-arbuckle@ouhsc.edu

Course Objective Review the biochemical processes most frequently tested on the NBDE

General Outline- 1st hour

Brief overview of Enzyme Kinetics

Enzyme Regulation and Inhibition Carbohydrate Structure and Function

enz (z) A B

Michaelis-Menton Kinetics
[substrate] vs. Vo is hyperbolic At high [substrate] the reaction reaches its maximum velocity (Vmax) The Km is the [substrate] at which the reaction velocity is Vmax The Km is an effective measure of affinity.

The Km is a Measure of Affinity

Km is inversely related to affinity

Enzymes with a low Km have a high affinity for substrate Enzymes with a high Km have a low affinity for substrate

Hexokinase vs Glucokinase
Hexokinase- Ubiquitously expressed High affinity for glucose Low Km and Vmax Saturated at low [glucose] Glucokinase- Liver specific enzyme High Km and Vmax Saturated at high [glucose]

Hexokinase functions during fasting Glucokinase functions after a glucose rich meal

Regulation of Enzyme Activity Allosteric Effectors

The activity of allosteric enzymes can be regulated by molecules that bind to sites on the enzyme other than the active site. Allosteric enzymes commonly contain multiple subunits and catalyze rate-limiting steps. Phosphofructokinase-1 (+) allosteric regulators: AMP, and Fructose, 2-6 bisphosphate (-) allosteric regulators: ATP and Citrate

Regulation of Enzyme Activity Covalent Modification

One of the primary means of modifying enzyme activity is by the addition or removal of a phosphate group. Phosphates are typically added by kinases and removed by phosphatases. Enzymes can either be activated or inactivated by the addition phosphate group. glycogen phosphorylase is active when phosphorylated glycogen synthase is inactive when phosphorylated

of a

Enzyme Inhibition
Competitive vs. Noncompetitive Competitive Inhibitors bind the same site on the enzyme as the substrate increase the Km of the enzyme do not affect the Vmax

Increasing the substrate concentration will eventually out-compete the inhibitor.

Competitive Inhibitors
Vmax is unchanged in the presence of a competitive inhibitor

Km is increased in the presence of a competitive inhibitor

Km is increased

Vmax is unchanged

Enzyme Inhibition
Competitive vs. Noncompetitive Noncompetitive Inhibitors do not bind the same site as the substrate do not affect the Km of the enzyme decreases the Vmax

The reaction will never reach the original maximum velocity, regardless of the substrate concentration

Noncompetitive Inhibitors
Vmax is decreased in the presence of noncompetitive inhibitor

Km is unchanged in the presence of a noncompetitive inhibitor

Km is unchanged

Vmax is decreased

Enzyme Inhibition and Regulation Takeaway Points

Competitive Inhibitors Increase the Km without affecting the Vmax Noncompetitive Inhibitors Do not affect the Km but decrease the Vmax Enzymes are regulated by allosteric effectors e.g. citrate is a negative regulator of PFK-1 covalent modification e.g. phosphorylation of glycogen phosphorylase activates the enzyme

Part II figure-3

Carbohydrates
General formula (CH2O)n Functions: Immediate Energy Source Blood Glucose Energy Stores Extracellular Matrix Glycoconjugates Nucleic Acid Precursor Glycogen (homopolymer) Glycosaminoglycans (heteropolymer) Glycoproteins Glycolipids Ribose

Carbohydrates
Disaccharides Lactose = Sucrose = Maltose = Polysaccharides Starchamylose (14 linkage) amylopectin, branched (14, 16) polysaccharides of glucose (16, 13, and 14) glucose + galactose glucose + fructose glucose + glucose

DextranLehninger Figure 1-10d

Structural Isomers Glucose Galactose Fructose

Product of oral bacteria Found in dental plaque

Glycosaminoglycans- Hyaluronic Acid Chondroitin Sulfate

Monosaccharides
Can be in an open chain form or a cyclic form In the open chain form, each has a carbonyl.

Six carbon sugars tend to be cyclic Cyclization generates an anomeric carbon isomers, and Lehninger Figure 7-6

two

Sugars with free anomeric carbons are reducing sugars

Disaccharides

Lactose = galactose(14) glucose Anomeric carbon for galactose is C-1 Anomeric carbon for glucose is C-1 C-1 of glucose remains free Lactose is a reducing sugar Reducing End of Lactose

Sucrose = fructose

glucose(12)

Anomeric carbon for glucose is C-1 Anomeric carbon for fructose is C-2 No free anomeric carbon Sucrose is not a reducing sugar Lehninger Figure 7-12

Starch

Storage form of glucose in plants Is made of amylose and amylopectin Amylose polymerized glucose no branches (14) linkage

(14)

Lehninger Figure 7-15

Amylopectin polymerized glucose branched (14) linkage (16) at branch points many reducing ends for quick mobilization

Glycogen
Primary linkage (14)

Branch point linkage (16)

Storage form of glucose in humans Primarily stored in liver functions to maintain blood glucose levels Stored in muscle functions to provide glucose during exercise

Lehninger Figure 7-9

Heteropolysaccharides Glycosaminoglycans
GAGs are long, unbranched heteropolysaccharide chains composed of repeating disaccharide units. Modified by negatively charged carboxyl and sulfate groups Extended, unbranched chains surrounded by a shell of water gel-like matrix or ground substance Neighboring molecules tend to repel each other slippery nature of mucous secretions and synovial fluid Compressible but rebound after the release of pressure resilience of synovial fluid and vitreous humor GAGs vary based on their disaccharide subunits, linkage, sulfation patterns and distribution in the human body. Majority are linked to a core protein to form a proteoglycan

Chondroitin Sulfate
Most abundant GAG in the body Found in cartilage, tendons, ligaments and aorta Forms proteoglycan aggregates with hyaluronic acid.

Hyaluronate/Hyaluronic Acid
Different from other GAGs unsulfated not covalently attached to proteins Is a lubricant and shock absorber Lehninger Figure 7-24 Found in synovial fluid, vitreous humor, loose connective tissue

Additional GAGs
Keratin Sulfate present in cornea and connective tissue Dermatan Sulfate found in skin, blood vessels and heart valves Heparin

the only intracellular GAG

found in mast cells lining arteries of liver, lungs and skin functions as an anticoagulant Heparin Sulfate extracellular found in basement membranes

Proteoglycans

All GAGs, except for hyaluronate, are covalently attached to proteins to form proteoglycans. In cartilage, a core protein is linked to linear GAG chains. The negative charge of GAGs causes repulsion between molecules, generating a bottle brush appearance

Proteoglycans
Sugar Content >> Protein Content

Mucopolysaccharidoses
GAGs are normally degraded in lysosomes by specific enzymes Deficiency in any of these enzymes leads to accumulation of GAGs and a variety of symptoms such as skeletal and extracellular matrix deformities and mental retardation Hurler Syndrome -L-Iduronidase deficiency sx- corneal clouding mental retardation dwarfing Iduronate sulfatase deficiency X-linked inheritance sx- NO corneal clouding physical deformity mental retardation

Hunter Syndrome

Glycoproteins
Proteins modified by oligosaccharides Differ from proteoglycans carbohydrate chain is shorter, branched, fewer negative charges Function in cell surface recognition cell surface antigenicity (e.g. blood group antigens) extracellular matrix mucins of GI/GU tracts (biological lubricants)

Glycoproteins (contd)
O-glycosidic bonds carbohydrate is attached to hydroxyl group of serine or threonine N-glycosidic bonds carbohydrate is attached to the amide group of an asparagine side chain Occur as post-translational modifications the

Carbohydrates Takeaway Points

Monosaccharides Disaccharides Polysaccharides Homopolysaccharides Heteropolysaccharides Starch Glycogen GAGs

Extended linear repetitions of disaccharides Central components of the extracellular matrix Hyaluronic Acid, Chondroitin Sulfate Glycoproteins Post-translational glycosylation of serine, threonine or asparagine residues Function in cellular recognition, cell surface antigenicity

Course Objective Review the biochemical processes most frequently tested on the NBDE

General Outline- 2nd hour Carbohydrate Metabolism Glycolysis Krebs Cycle Quiz

Digestion of Carbohydrates
Begins in the mouth - amylase breaks (14) bonds of starch and glycogen oligosaccharides In the small intestine - amylase produced by the pancreas further digests starch and glycogen oligo- and di-saccharidases made by intestinal mucosal cells catalyze the final digestion Small intestine absorbs

monosaccharides

Major Pathways of Glucose Utilization

Glycolysis
Anaerobic Cytosolic

Two phases Preparatory (requires energy ) Pay-off (produces energy)

Kinase: Isomerase: Aldolase:

Phosphorylates Rearranges Cleaves C-C figure-14-2a

- 2 ATP per glucose

Glycolysis
Anaerobic Cytosolic
Two phases Preparatory - 2 ATP per glucose Pay-off + 4 ATP per glucose Net 2 ATP per glucose

+ 4 ATP per glucose

figure-14-2b

Glucose Glucose-6-Phosphate
ATP ADP

Regulation of Glycolysis
Phosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of Pyruvate
Phosphorylation of Glucose commits glucose to inside cell two isoforms (Hexokinase vs Glucokinase) hormonal regulation

Hexokinase vs Glucokinase
Hexokinase- Ubiquitously expressed High affinity for glucose Low Km and Vmax Saturated at low [glucose] Glucokinase- Liver specific enzyme High Km and Vmax Saturated at high [glucose]

Liver has a selective advantage for the uptake of glucose

Regulation of Glycolysis
Phosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of Pyruvate

Phosphofructokinase-1
Rate-Limiting Step Commits Glucose to Glycolysis Allosterically Regulated (-) ATP, citrate (+) AMP, F2,6BP

Fructose-6-Phosphate
ATP

ADP

Fructose-1,6-Bisphosphate

Hormonally Regulated

Regulation of Glycolysis
Phosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of Pyruvate

Pyruvate Kinase
Feed-forward Regulation (+) F1,6,BP Large (-) G Essentially Irreversible Hormonally Regulated

Phosphoenolpyruvate
ADP

ATP

Pyruvate

Hormonal Regulation of Glycolysis

Insulin Favors Glycolysis Upregulates the synthesis of Hexokinase Phosphofructokinase Pyruvate Kinase Glucagon Opposes Glycolysis Downregulates the synthesis of Hexokinase Phosphofructokinase Pyruvate Kinase

Glycolysis Takeaway Points

Anaerobic process Occurs exclusively in the cytosol Glucose (a six-carbon molecule) is degraded to 2 molecules of pyruvate (a three carbon molecule) Important sites of regulation phosphorylation of glucose phosphorylation of fructose-6-phosphate generation of pyruvate Energy Produced Per Glucose Molecule 2 NADH 2 ATP hexokinase PFK-1 Pyruvate Kinase

Fates of Pyruvate

Anaerobic Cytosolic

Lehninger Figure 14-3

Anaerobic Conditions

Pyruvate is converted to lactate In the absence of oxygen, glycolysis is the only means of generating ATP. Regenerates the NAD+ required by glyceraldehyde-3-phosphate dehydrogenase during the payoff phase of glycolysis Anaerobic bacteria also rely on this pathway to maintain ATP production

Lehninger Figure p538

Aerobic Conditions

Anaerobic Conditions

TCA Cycle

Pyruvate Dehydrogenase

Lehninger Figure 16-2

Converts pyruvate to CO2 and Acetyl CoA TCA cycle Multi-subunit enzyme requires several cofactors A Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH Negative allosteric regulation by Acetyl CoA and NADH

(mitochondrial matrix)

Lehninger Figure 16-7

-Ketoglutarate Dehydrogenase

Lehninger Figure page 610

Analogous to Pyruvate Dehydrogenase Multi-subunit enzyme requires several cofactorsA Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH

TCA Cycle
Occurs in the mitochondrial matrix Citrate synthase is the rate-limiting step Sites of NADH synthesis Isocitrate dehydrogenase - Ketoglutarate dehydrogenase Malate dehydrogenase Site of FADH2 synthesis Succinate dehydrogenase (member of the ETC) Site of GTP synthesis Succinyl-CoA synthetase

Regulation of the TCA Cycle

Negative Regulators Indicative of high energy state ATP NADH Acetyl-CoA Citrate Succinyl-CoA Positive Regulators Indicative of low energy state AMP/ADP CoA NAD+ Ca2+
Lehninger Figure 16-18

TCA Cycle
For each Acetyl CoA 2 CO2 1GTP 3 NADH 1 FADH2

ETC

Lehninger Figure 16-7

ATP Produced
by both substrate level phosphorylation and oxidative phosphorylation

Energy Producing Reaction 3 NADH 3 NAD+ FADH2 GDP + Pi GTP

Number of ATP produced 9 2 1

12 ATP/ Acetyl CoA oxidized

Catabolism of Macromolecules
Converges on the formation of Acetyl CoA

Krebs Cycle Takeaway Points

When oxygen is available Pyruvate Acetyl CoA
(not technically a part of the TCA cycle)

Occurs exclusively in the mitochondrial matrix None of the reaction intermediates are consumed For each Acetyl CoA oxidized to 2 CO2 3 NADH 1 FADH2 1 GTP Reduced equivalents will be passed to the ETC for ATP synthesis Cycle is inhibited by high-energy substrates

Biochemistry Review for the NBDE 2008

November 7, 2008
General Outline Carbohydrate Metabolism Review Oxidative Phosphorylation Insulin Signaling Glucagon Signaling
Contact Information: janeen-arbuckle@ouhsc.edu

Glycolysis Takeaway Points

Anaerobic process Occurs exclusively in the cytosol Glucose (a six-carbon molecule) is degraded to 2 molecules of pyruvate (a three carbon molecule) Important sites of regulation phosphorylation of glucose phosphorylation of fructose-6-phosphate generation of pyruvate Energy Produced Per Glucose Molecule 2 NADH 2 ATP hexokinase PFK-1 Pyruvate Kinase

Aerobic Conditions

Anaerobic Conditions

TCA Cycle

Pyruvate Dehydrogenase

Lehninger Figure 16-2

Converts pyruvate to CO2 and Acetyl CoA TCA cycle Multi-subunit enzyme requires several cofactors A Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH Negative allosteric regulation by Acetyl CoA and NADH

TCA Cycle
Occurs in the mitochondrial matrix Citrate synthase is the rate-limiting step Sites of NADH synthesis Isocitrate dehydrogenase - Ketoglutarate dehydrogenase Malate dehydrogenase Site of FADH2 synthesis Succinate dehydrogenase (member of the ETC) Site of GTP synthesis Succinyl-CoA synthetase

TCA Cycle
For each Acetyl CoA 2 CO2 1GTP 3 NADH 1 FADH2

ETC

Lehninger Figure 16-7

Electron Transfer and Oxidative Phosphorylation

Glucose metabolism thus far: 4 ATP 10 NADH 2 FADH2 ETC oxidizes reduced cofactors ATP Occurs in the inner mitochondrial membrane Hydrogen ions are pumped to the intermembrane space gradient Hydrogen ions drive ATP synthesis
figure-16-1

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm

Flow of Electrons Complex I Complex II Complex III Complex IV NADH dehydrogenase Succinate dehydrogenase Cytochrome bc1 complex Cytochrome oxidase (a+a3)

Coenzyme Q (CoQ) (also called ubiquinone) Cytochrome c (Cyt c) Complex V ATPase

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm

H+

H+

H+

e-

e-

e-

eH 2O

2H++ O2
NADH + H+ NAD+

NADH is oxidized by Complex I Electrons are passed down the chain H+ are pumped to the intermembrane space Only complexes I, III, and IV pump H+ Three H+ are pumped/NADH Oxygen is the terminal electron acceptor

H+

H+

e-

e-

eH 2O

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm 2 2H++ O

FADH2+

FAD

FADH2 is oxidized by Complex II Complex II is Succinate Dehydrogenase Electrons are passed down the chain H+ are pumped to the intermembrane space Two H+ are pumped per FADH2

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm

H+ ee-

H+ ee-

H+

H+

H+
ATPase

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htmO2 2H++

H 2O

NADH + H+

NAD+

Electron transfers generates a H+ gradient The energy generated by this gradient drives the synthesis of ATP

ADP + Pi

ATP

1 NADH 3 ATP 1 FADH2 2 ATP

Oxidative Phosphorylation Takeaway Points

The electron transport chain is located in the inner mitochondrial matrix. NADH and FADH2 derived from the catabolism of glucose (and other macromolecules) donate electrons to the chain Oxygen is the terminal electron acceptor Hydrogen ions are pumped to the intermembrane space electrical gradient and pH gradient The flow of H+ back into the mitochondrial matrix powers the synthesis of ATP by the ATPase

Insulin Signaling
Elevated blood glucose cells of the pancreas Insulin Insulin signaling increases expression of GLUT-4 receptors in insulin responsive tissues Skeletal muscle Glycogen Adipose tissue Triacylglycerol (TAG) Insulin promotes Glucose Uptake Glycolysis Glycogen Synthesis HMP shunt Triacylglycerol Synthesis Insulin signal Dephosphorylation of enzymes Enzymes activated by insulin are dephosphorylated in the active state

Structure of Insulin

Synthesized as Preproinsulin (RER) Loses its signal sequence to become Proinsulin Disulfide bonds link Chain A and Chain B (RER) Loses its C-peptide to become Insulin (Golgi)

Insulin Receptor
Cell-surface receptor Contains intrinsic tyrosine kinase activity Autophosphorylates Activates kinases and phosphatases Insulins effects are mediated by dephosphorylation
Lehninger Figure 12-6

Enzymes activated by insulin are active in dephosphorylated form Glycogen Synthase

Glucagon cAMP Protein Kinase A ATP ADP

Insulin Signaling
Increases Glucose uptake Glycogen Synthesis Protein Synthesis Fat Synthesis Decreases Gluconeogenesis Glycogenolysis Lipolysis Activates/Deactivates Enzymes
Alters Gene Expression Increases Glucose Transport

Glycogen Synthase a (active) Pi

P Glycogen Synthase b (inactive)

H 2O

Protein phosphatase

Insulin

Insulin
Activates/Deactivates Enzymes

Alters Gene Expression

Increases Glucose Transport

Insulin
Activates/Deactivates Enzymes Alters Gene Expression

Increases Glucose Transport

Lehninger Box 11-02 Figure 1

Insulin Responsive Tissues GLUT- 4 Surface Expression

Insulin
Activates/Deactivates Enzymes Alters Gene Expression Increases Glucose Transport

Lehninger Figure 15-36

Insulin Takeaway Points

Peptide hormone Receptor has intrinsic tyrosine kinase activity

Glucose uptake Glycogen Synthesis Protein Synthesis Fat Synthesis Gluconeogenesis Glycogenolysis Lipolysis
Alters enzyme activity Activates Glycogen Synthase Alters gene expression Upregulates genes of glycolysis Promotes GLUT-4 translocation skeletal muscle & adipose tissue

Glucagon Signaling

Low blood glucose cells of the pancreas Glucagon Promotes the mobilization of blood glucose glycogenolysis gluconeogenesis Glucagon promotes protein and/or fat catabolism Glucagon signal phosphorylation of enzymes Enzymes activated by glucagon are phosphorylated in the active state Actions of Glucagon can also be performed by Epinephrine

Glucagon/Epinephrine Signaling

Lehninger Figure 12-12

Activates G protein Activates Adenylate Cyclase cAMP Activates cAMP dependent protein kinase, Protein Kinase A

Glucagon/Epinephrine Signaling Activates a G protein Heterotrimeric () Inactive (GDP ) Active GTP

Lehninger Figure 12-14

()

Activates Adenylate Cyclase

Glucagon/Epinephrine Signaling
Leads to the phosphorylation of enzymes Phosphorylation activates glycogen phosphorylase a Glycogenolysis

Phosphorylation inactivates glycogen synthase Halts Glycogen Synthesis

Lehninger Figure-15-25

Glucagon
cAMP

Glucagon/Epinephrine Signaling

Protein Kinase A
ATP ADP

Glycogen Synthase a (active) Pi

P Glycogen Synthase b (inactive)

Decreases Glycogen Synthesis

H 2O

Protein phosphatase

Increases Glycogenolysis Gluconeogenesis Ketogenesis Uptake of amino acids

Insulin

Glucagon
cAMP Protein Kinase A
ATP ADP

Glycogen is Degraded
Glycogen P Phosphorylase a (active)
ADP ATP H 2O Pi Insulin Protein phosphatase

Glycogen Phosphorylase Kinase b (inactive)

Pi

P Glycogen Phosphorylase Kinase a (active)

H 2O

Protein phosphatase

Glycogen Phosphorylase b (inactive)

Insulin

Glucagon Takeaway Points

Peptide hormone Receptor is associated with G-protein Activates Adenylate Cyclase cAMP (the second messenger)

Glycogenolysis Gluconeogenesis Ketogenesis Uptake of amino acids Glycogenesis

cAMP activates Protein Kinase A physiological response is due to the phosphorylation of enzymes Enzymes activated by Glucagon are active when phosphorylated Glycogen Phosphorylase Kinase Glycogen Phosphorylase

Biochemistry Review for the NBDE 2008

November 12, 2008
General Outline- 2nd hour Carbohydrate Metabolism Glycogen Synthesis Hexose Monophosphate Shunt Glucose Mobilization Glycogenolysis Gluconeogenesis
Contact Information: janeen-arbuckle@ouhsc.edu

Major Pathways of Glucose Utilization Insulin Favors Glucagon Opposes

Glycogen Synthesis

Skeletal muscle and liver take up glucose and store it as glycogen [(14) and (16)] Glycogen synthesis occurs during the well-fed state Insulin favors glycogen synthesis Glucagon opposes glycogen synthesis

Glycogen Synthesis
glucose 6- phosphate glucose 1- phosphate
phosphoglucomutase

glucose 1- phosphate + UTP UDP-glucose + PPi

UDP-glucose pyrophosphorylase

UDP- glucose is the substrate for glycogen synthesis Anomeric carbon of sugar is activated by attachment to UDP

Insulin favors glycogen synthesis

Lehninger Page 565

Glucagon opposes glycogen synthesis

Glycogen Synthesis Glycogen Synthesis

+ +

Lehninger Figure 15-8

Lehninger Figure 15-9

Glycogen Synthesis
Two enzymes glycogen synthase glycogen-branching enzyme

Synthase activated by insulin inactivated by glucagon

Glucagon
cAMP

Glucagon Signals through the activation of Protein Kinase A Glycogen Synthase is inactive in the phosphorylated form

Protein Kinase A
ATP ADP

Glycogen Synthase a (active) Pi

P Glycogen Synthase b (inactive)

H 2O

Protein phosphatase

Insulin Signals through the activation of Protein Phosphatase Glycogen Synthase is active in the dephosphorylated form

Insulin

Glycogen Synthesis Takeaway Points

Occurs in skeletal muscle and liver [(14), (16)] Occurs during the well-fed state Substrate is UDP-glucose added to the non-reducing end of a growing glycogen chain Important enzymes Glycogen Synthase Glycogen Branching Enzyme Glycogen synthase is active in the dephosphorylated form (promoted by insulin) Glycogen synthase is inactive in the phosphorylated form (promoted by glucagon)

Insulin favors glycogen synthesis Glucagon opposes glycogen synthesis

Major Pathways of Glucose Utilization Insulin Favors Glucagon Opposes

Pentose Phosphate Pathway

Also referred to as Hexose Monophosphate Shunt 6-phosphogluconate Pathway Products Ribose 5-Phosphate NADPH Site of Regulation Glucose 6-Phosphate Dehydrogenase (G6PD)

Lehninger Figure 14-20

Oxidative Phase of Pentose Phosphate Pathway

Glucose 6-phosphate
Glucose 6-Phosphate Dehydrogenase NADP+

NADPH

6-Phosphogluconate
6-Phosphogluconate Dehydrogenase NADP+

NADPH

Ribose 5-phosphate + CO2

Products of the Pentose Phosphate Pathway NADPH Fatty Acid and Cholesterol Synthesis
Counter Free Radicals RBCs are exposed to oxygen generated free radicals Glutathione functions to eliminate these free radicals NADPH is required to keep glutathione in the reduced state G6PD deficiency free radical damage hemolytic anemia
Lehninger Box-14-03

Nonoxidative Phase of Pentose Phosphate Pathway

Lehninger Figure 14-22

Occurs in cells not needing the pentose sugars Functions to regenerate glucose 6-phosphate

Glucose Utilization Take Away Points

Glucose is the bodys preferred substrate Insulin levels increase in response to elevated blood glucose Increase glucose uptake by skeletal muscle and adipose tissue Stimulates the phosphorylation of glucose Glucose
ATP Hexokinase ADP

Glucose 6-Phosphate Glucose 6-Phosphate can be used in Glycolysis Glycogen Synthesis HMP Shunt

General Outline- 2nd hour Carbohydrate Metabolism Glycogen Synthesis Hexose Monophosphate Shunt Glucose Mobilization Glycogenolysis Gluconeogenesis

Maintenance of Blood Glucose

Blood glucose levels drop quickly after a meal

Glucagon:Insulin Ratio
Activates two processes to maintain blood glucose Glycogenolysis skeletal muscle liver Gluconeogenesis liver kidney

Glycogenolysis
Glycogenolysis functions during the first 24 hours of fasting Glucose from liver glycogen blood glucose Glucose from muscle glycogen energy for the myocyte

Glucagon favors glycogenolysis Insulin opposes glycogenolysis

Glycogenolysis

Lehninger Figure 15-3

Glucagon Activates

Insulin Inactivates

Glycogenolysis

Insulin

Glucagon activates Phosphorylase a by phosphorylating it [activates Phosphorylase b Kinase]

Lehninger Figure 15-24

Insulin inactivates Phosphorylase a by dephosphorylating it [activates phosphorylase a phosphatase]

Glucagon
cAMP Protein Kinase A
ATP ADP

Glycogen is Degraded
Glycogen P Phosphorylase a (active)
ADP ATP H 2O Pi Insulin Protein phosphatase

Glycogen Phosphorylase Kinase b (inactive)

Pi

P Glycogen Phosphorylase Kinase a (active)

H 2O

Protein phosphatase

Glycogen Phosphorylase b (inactive)

Insulin

Glycogenolysis
Glucose 1-phosphate>>Glucose
Activated by Glucagon

Glucose 1-Phosphate Glucose 6-Phosphate Glucose 6-phosphatase

only in the liver and kidney

Glucose from liver glycogen blood glucose Glucose from muscle glycogen energy for the myocyte Lehninger Figure 15-4

Glycogenolysis Take Away Points

Glycogen is mobilized within four hours of eating Stimulated by Glucagon phosphorylates and activates glycogen phosphorylase kinase glycogen phosphorylase phosphorylates and inactivates glycogen synthase Occurs in liver blood glucose and muscle energy

Maintenance of Blood Glucose

Blood glucose levels drop quickly after a meal

Glucagon:Insulin Ratio
Activates two processes to maintain blood glucose Glycogenolysis skeletal muscle liver

Gluconeogenesis liver kidney

Gluconeogenesis
The de novo synthesis of glucose Occurs in the liver>>kidney during fasting Substrates include all intermediates of glycolysis all intermediates of the TCA cycle lactate glycerol -ketoacids of glucogenic amino acids Is NOT a reversal of Glycolysis Must bypass Hexokinase Phosphofructokinase Pyruvate Kinase

Acetyl CoA is NOT a substrate for gluconeogenesis

Irreversible Steps in Glycolysis

Three important sites of regulation Large G Reactions are Irreversible

Gluconeogenesis in NOT a Reversal of Glycolysis

To counter the irreversible steps of glycolysis Four enzymatic steps Four ATP and Two GTP

Enzymes in Glycolysis
Hexokinase

Enzymes in Gluconeogenesis
Glucose-6-Phosphatase Fructose 1,6-bisphophatase Phosphoenolpyruvate Carboxykinase Pyruvate Carboxylase

Phosphofructokinase

Pyruvate Kinase

Conversion of Pyruvate to Phosphoenolpyruvate

Requires Two Enzymes Pyruvate Carboxylase Phosphoenolpyruvate (PEP) Carboxykinase
Pyruvate Carboxylase (mitochondrial matrix) Pyruvate (3C) is Carboxylated to Oxaloacetate (4C) Requires Biotin, ATP, and CO2 Oxaloacetate can not cross the mitochondrial membrane so it is converted to malate PEP Carboxykinase (cytosol) Oxaloacetate (4C) is decarboxylated and phosphorylated to form PEP (3C) Requires GTP

Mitochondrion
Pyruvate + CO2
ATP
Acetyl CoA

ADP + Pi

Pyruvate Carboxylase + Biotin

Oxaloacetate

Malate

Cytosol

Malate

Oxaloacetate
GTP PEP Carboxykinase GDP

Phosphoenolpyruvate + CO2

PEP

Reverse Glycolysis

Fructose 6-Phosphate

Conversion of Fructose 1,6-bisphosphate to Fructose 6-Phosphate

PEP Reverse Glycolysis Fructose 6-Phosphate

Fructose 1,6- bisphosphate

H 2O Fructose 1,6bisphosphatase P

Bypasses the irreversible phosphofructokinase-1 reaction Stimulated by High ATP Low AMP Inhibited by Elevated AMP Fructose 2,6-bisphosphate

Fructose 6 - phosphate

Conversion of Glucose 6-Phosphate to Glucose

Fructose 6-Phosphate Glucose 6-Phosphate Glucose

Bypasses the irreversible hexokinase reaction Only occurs in the LIVER and KIDNEY Glucose 6-phosphatase is essential for glucose to be released into the blood stream Without this enzyme, glucose generated by glycogenolysis and gluconeogenesis can not be released to the body

Substrates for Gluconeogenesis All intermediates of glycolysis All intermediates of the TCA cycle Lactate Glycerol -Ketoacids of glucogenic amino acids
When glucose is limiting Muscles go into anaerobic metabolism Lactate Fats are catabolized Glycerol and fatty acids Proteins are catabolized amino acids

Acetyl CoA is NOT a substrate for gluconeogenesis

glucose lactate glucose

Cori Cycle
Active muscle mobilizes glycogen stores glucose Glucose is metabolized by glycolysis under anaerobic conditions lactate Lactate enters blood liver Gluconeogenesis resynthesizes glucose from lactate Glucose enters blood muscle Lehninger Figure 23-18 Gluconeogenesis occurs in LIVER and KIDNEY Glucose can be metabolized or stored as muscle glycogen

Substrates for Gluconeogenesis

Lysine and Leucine are NOT glucogenic

Regulation of Gluconeogenesis
Glucagon Phosphorylates enzymes deactivation Pyruvate Kinase PFK-2 Induces the expression of PEP carboxykinase Substrate Availability Glucogenic amino acids increase rate Reciprocal Control + Acetyl CoA Activates pyruvate carboxylase Inhibits pyruvate dehydrogenase Inhibits Fructose 1,6-bisphosphatase - AMP Activates PFK-1

Gluconeogenesis Takeaway Points

Occurs in liver and kidney Is NOT a reversal of Glycolysis Unique Enzymes Glucose 6-Phosphatase Fructose 1,6-Bisphosphatase PEP Carboxykinase Pyruvate Carboxylase (Biotin) Substrates include Glycolysis and TCA intermediates Glucogenic amino acids Glycerol Lactate Cori Cycle Lactate returns to the liver glucose

Lehninger Figure 14-16

General Outline- 2nd hour Lipid Structure and Function Cholesterol and Fatty Acids Compartmental Barrier Derivatives of Cholesterol and FAs Fatty Acid Synthesis

Cholesterol
Abundant in cell membrane Also functions as a precursor Bile acids Vitamin D Steroid hormones Transported in Lipoprotein Particles Most abundant in LDL>HDL>VLDL Found in the diet and synthesized in all tissues Especially liver, adrenal cortex and reproductive tissues
Lehninger Figure-10-16

Rate limiting step is catalyzed by HMG-CoA reductase Synthesis requires NADPH

Fatty Acids (FA)

Lehninger Figure-10-1

Amphipathic Hydrophobic AND Hydrophilic Saturated Unsaturated Bonds No Double Bonds One Double

>90% are esterified Cholesteryl esters Phospholipids Triacylglycerides (TAG) Saturated Unsaturated Free FAs circulate bound to ALBUMIN ( during fasting)

Function Oxidized for energy by most tissues (esp. liver and muscle) Structural roles (phospholipids & glycolipids) Precursor of prostaglandin Major Energy Store (TAG)

Unsaturated Saturated

Essential

Triacylglycerol (TAG)

May also be referred to as triglycerides Major energy store found in adipocytes Three fatty acids esterified to glycerol Neutral Fat When mobilized FA Acetyl CoA TCA Glycerol glycolysis or gluconeogenesis
Lehninger Figure-10-2

Cholesterol and Fatty Acids From Diet or Synthesized de novo

Lehninger Figure-17-1

Chylomicrons

Largest, least dense lipoprotein Predominantly dietary triacylglycerol Generated in enterocytes lymph blood stream tissues Tissues will consume or store the dietary triacylglycerol Remnants return to the liver release their cholesterol degraded in lysosomes

Lehninger Figure-17-2

Lehninger Figure 21-40

Lipoprotein Particles
Chylomicrons Transport TAG from the diet to the tissues Apo B-48 LDL Particles Transport cholesterol to the tissues Receptor-mediated endocytosis Apo B-100 VLDL Particles Transport TAG synthesized by the liver to the tissues HDL Particles Transport cholesterol to the liver for elimination

Fatty Acid and Lipoprotein Particle Take Away Points

Fatty acids part of phospholipids and glycolipids stored as neutral fat (TAG) free fatty acids are transported bound to albumin From the diet or synthesized from excess carbohydrates and proteins in the diet Saturated no double bonds Unsaturated double bonds palmitoleic, oleic linoleic, linolenic, arachidonic acid Chylomicrons VLDL LDL HDL Dietary TAG to the tissues Hepatic TAG to the tissues Cholesterol to the tissues Cholesterol to the liver for elimination

Lehninger Figure-10-6

Glycerophospholipids Lecithin Sphingolipids Sphingomyelin

Glycerophospholipids
General Structure Glycerol backbone Two Fatty Acids Phosphate Group + Polar Head Group

Know Lecithin (Phosphatidylcholine) glycerol phorphoric acid fatty acids choline

(one of the most abundant phospholipids in cells)
Lehninger Figure-10-8

Sphingolipids

General Structure Sphingosine backbone One Fatty Acid Head Group Sphingomyelin Sphingolipid prevalent in myelin Also contains choline Deficiency in degradation Sphingolipidoses
Lehninger Figure-10-12

Sphingolipidoses
Tay-Sachs Hexosaminidase A deficiency GM2 gangliosides accumulate Neurodegeneration Cherry-red Macula Gauchers Glucocerebrosidase deficiency Glucocerbrosides accumulate Hepatosplenomegaly Osteoporosis of long bones Niemann-Pick disease Sphingomyelinase deficiency Spingomyelin accumulation Hepatosplenomegaly Neurodegenerative

Lehninger Box 10-2 Figure-1

Phospholipid Membrane as a Barrier

Small, non-polar molecules enter cells by Simple Diffusion Down gradient without a transporter
STEROID HORMONES Circulate bound to a protein Diffuse through the membrane Have an INTRAcellular receptor (cytosolic or nuclear) Ultimately reach the nucleus Alters gene expression

Phospholipid Membrane as a Barrier

Large, polar substances require transporters to cross membranes Finite number of transporters saturation kinetics

Facilitated diffusion occurs down gradient does not require energy e.g. glucose uptake Active transport against gradient requires energy e.g. Na/K ATPase

Phospholipid Membrane Takeaway Points

Amphipathic Phospholipids and Glycolipids form lipid bilayer Know Lecithin and Sphingomyelin Cholesterol is abundant Membrane functions as a barrier polar substances require a transporter glucose or cell surface receptor peptide hormones nonpolar substances diffuse across membrane steroids

Three forms of transport simple diffusion facilitated diffusion require transporter active transport

down gradient

Steroid Hormones
Derived from cholesterol Nonpolar, lipophilic Circulate bound to a carrier protein Enter cells by simple diffusion Bind to INTRAcellular receptors Ultimately reach the nucleus to alter gene expression
Lehninger Figure-10-19

Response to steroid hormone stimulation is slow

Eicosanoids
Prostaglandins, Thromboxanes, Leukotrienes
Have Hormone-Like Properties Physiological and Pathological Responses Vasoconstriction/Vasodilation Contraction/Relaxation of smooth muscle Bronchoconstriction and Anaphylaxis (Leukotrienes) Differences From Hormones Act locally Not stored Short half-life Produced by all tissues in small amounts Derived from arachidonic acid, a fatty acid component of the cell membrane

Lehninger Figure-10-18

Arachidonic Acid liberated from phospholipid by Phospholipase A2 Converted by Cyclooxygenase to Prostaglandins and/or Thromboxanes Converted by Lipooxygenase to Leukotrienes

de novo Fatty Acids Synthesis

A large portion of fatty acids come from the diet chylomicrons tissues Fatty acids can also be synthesized from carbohydrates and proteins Primarily occurs in the liver and mammary glands, and, to a lesser extent, in adipose tissue Requires Acetyl CoA, ATP, Biotin, and NADPH Fatty Acid Synthesis Occurs in the Well-Fed State High Insulin:Glucagon Ratio

de novo Fatty Acids Synthesis

Step One Transfer of Acetyl CoA across mitochondrial membrane
The Coenzyme A portion of the molecule can not cross the membrane Acetyl CoA condenses with Oxaloacetate to form Citrate Citrate crosses the membrane and is converted back to Acetyl CoA and Oxaloacetate

Mitochondrion
Oxaloacetate + Acetyl CoA
Citrate Synthase

CoA

Citrate

Cytosol
CoA, ATP

Citrate
ATP-Citrate Lyase

ADP + Pi

Oxaloacetate + Acetyl CoA

de novo Fatty Acids Synthesis

Step One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoA
Occurs in the cytosol Catalyzed by Acetyl CoA carboxylase Rate-Limiting Step Allosteric Regulation and Covalent Modification

Allosteric Regulation of Acetyl CoA Carboxylase

Rate-limiting enzyme in de novo fatty acid synthesis Activated by Citrate, an indicator of high energy state Inhibited by Long-chain Fatty AcylCoA, the end-product Acetyl CoA + CO2
ATP

LCFA-CoA

ADP + Pi

Acetyl CoA Carboxylase + Biotin

Citrate

Malonyl CoA

Glucagon
cAMP

Covalent Modification of Acetyl CoA Carboxylase

Glucagon Signals through the activation of Protein Kinase A Acetyl CoA Carboxylase is inactive in the phosphorylated form Insulin Signals through the activation of Protein Phosphatase Acetyl CoA Carboxylase is active in the dephosphorylated form

Protein Kinase A
ATP ADP

Acetyl CoA Carboxylase (active) Pi

P Acetyl CoA Carboxylase (inactive)

H 2O

Protein phosphatase

Insulin

de novo Fatty Acids Synthesis

Step One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoA Step Three Elongation of fatty acid chain by Fatty Acid Synthase
Utilizes additional subunits of Acetyl CoA and NADPH

de novo Fatty Acids Synthesis

Step One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoA Step Three Elongation of fatty acid chain by Fatty Acid Synthase Step Four Esterification of FAs to Glycerol TAG

de novo Fatty Acids Synthesis Takeaway Points

Fatty Acid Synthesis Occurs in the Well-Fed State High Insulin:Glucagon Ratio The rate limiting step is catalyzed by Acetyl CoA Carboxylase Allosteric regulation Covalent modification Predominantly occurs in the liver and mammary glands Requires Acetyl CoA, ATP, Biotin, and NADPH Occurs in the cytosol

Biochemistry Review for the NBDE 2008

November 19, 2008
General Outline- 1st hour Fatty Acid Catabolism Ketone Synthesis Protein Metabolism Production of Ammonia Urea Cycle Metabolism of -Ketoacids
Contact Information: janeen-arbuckle@ouhsc.edu

Epinephrine
cAMP

Fatty Acids Mobilization and Oxidation

TAGs are the bodys main energy store Concentrated energy source highly reduced and anhydrous To obtain energy TAG glycerol three free FAs Hormone-Sensitive Lipase Active when phosphorylated glucagon/epinephrine Inactive when dephosphorylated insulin

Protein Kinase A
ATP ADP

HormoneSensitive Lipase (inactive) Pi

Hormone- P Sensitive Lipase (active) H 2O

Protein phosphatase

Insulin

Triacylglycerol

HormoneSensitive Lipase

Fatty Acids Mobilization and Oxidation

Glycerol + Free Fatty Acids BLOOD

Tissues

DHAP

Mitochondria

-Oxidation

Acetyl CoA
Brain and RBCs can NOT use FAs

TCA

 -oxidation of Fatty Acids

Occurs in the Mitochondria Fatty Acids must use the Carnitine Shuttle Carnitine Acyl Transferase is inhibited by Malonyl CoA

Lehninger Figure-17-6

 -oxidation of Fatty Acids

Occurs during prolonged fasting Occurs in the mitochondria of liver and muscle Oxidizes fatty acids to Acetyl CoA FADH2 NADH TCA

ATP
Lehninger Figure-17-7

ETC

Fatty Acid Synthesis vs -Oxidation

Lehninger Figure-17-12

Glucagon and Insulin also regulate Hormone-Sensitive Lipase Active when phosphorylated by Glucagon Inactive when dephosphorylated by Insulin

Synthesis
Greatest flux through pathway Hormonal State favoring pathway Major tissue site Subcellular location Carriers of acyl groups Oxidation/reduction cofactors Two-carbon donor/ product Activator Inhibitor Product of Pathway After carbohydrate rich meal High Insulin:Glucagon Liver Cytosol Citrate NADPH Malonyl CoA (donates acyl) Citrate LCFA-CoA Palmitate

Degradation
In starvation Low Insulin:Glucagon Muscle, Liver Mitochondria Carnitine NAD+, FAD Acetyl CoA (product) Malonyl CoA Acetyl CoA

Well-Fed State Insulin Signaling

Abundant Glucose Glycolysis Glycogen Synthesis HMP Shunt Excess Glucose and/or protein de novo Fatty Acid Synthesis

Fasting/Starvation Glucagon/Epinephrine Signaling

Limited Glucose Glycogenolysis Gluconeogenesis Beta-oxidation of Fatty Acids Acetyl CoA Protein Catabolism

Ketone Bodies Acetoacetate, Acetone -hydroxybutyrate

Synthesized in liver mitochondria HMG-CoA synthase is rate-limiting Acetoacetate and -hydroxybutyrate blood Acetyl CoA TCA Important sources of energy Soluble in aqueous environment Produced in excess of livers need Used in proportion to their concentration in the blood Ketone bodies are used by the brain RBCs can NOT use ketone bodies
Lehninger Figure-17-18

Ketone Bodies Acetoacetate, Acetone -hydroxybutyrate

Formed during fasting and in Diabetes Type I Production in excess of rate of use Ketonemia Ketonuria

pKa value is approximately 4 Ketone dissociates, Release of hydrogen Lowers pH

Ketoacidosis

Fatty Acids Mobilization and Oxidation Takeaway Points

Triacylglycerol is the major form of energy storage Hormone sensitive lipase breaks TAG glycerol and FFAs Energy is derived from fatty acids by -oxidation Occurs in the mitochondria Degrades fatty acids to Acetyl CoA, FADH2, and NADH

-oxidation does NOT occur in the Brain or RBCs

In prolonged starvation Acetyl-CoA accumulates Ketones Rate-limiting step HMG CoA synthase Ketone Bodies= Acetoacetate, Acetone, -hydroxybutyrate

Brain can utilize Ketone Bodies RBCs can NOT use Ketone bodies

Protein Metabolism
Protein catabolism occurs in three scenarios after a protein-rich meal during normal turnover of proteins during prolonged fasting or starvation Proteins catabolism results in the generation of free amino acids. Amino acids are degraded to carbon skeletons substrate for glucose ammonia toxic byproduct that is eliminated

Lehninger Figure-18-3

Denaturation of Proteins

Lehninger Figure 3-16

Loss of higher order structure No hydrolysis of peptide bonds

Digestive Proteases
Stomach Parietal Cells HCl, Intrinsic Factor Chief Cells Pepsinogen Pepsin Pancreas Trypsinogen Chymotrypsinogen Procarboxypeptidases Intestine Enteropeptidase (Enterokinase) Cleaves trypsinogen to active trypsin Trypsin cleaves and activates itself and all other zymogens

Digestive Proteases

Trypsin (Serine Protease) Cleaves C-terminal to basic amino acids Chymotrypsin Cleaves C-terminal to aromatic amino acids Carboxypeptidases Remove the C-terminal residue of a peptide Aminopeptidases Remove the N-terminal residue of a peptide

Catabolism of Dietary Proteins

Amino acids are absorbed from the small intestine and enter the portal circulation The portal vein feeds the liver where amino acids are further catabolized Catabolism of amino acids requires two enzymes Transaminase (aminotransferase) Glutamate Dehydrogenase Amino acids are degraded to carbon skeletons (-ketoacids) ammonia (toxic byproduct)

Aminotransferase
Catalyzes the first step in amino acid catabolism Transfers amino group from amino acids to -ketoglutarate Glutamate and the -keto-acid of the amino acid degraded are formed Requires pyridoxal phosphate as a cofactor (from Vitamin B6) Nearly all amino acids funnel their amino groups to glutamate
Lehninger Figure-18-4

Amino Acid + -Ketoglutarate

Glutamate + -Keto acid

Glutamate Dehydrogenase
Catalyzes the second step in amino acid catabolism Only enzyme that can use either NAD or NADP Generates NH4+ and -Ketoglutarate

Lehninger Figure-18-7

Glutamate

NH4+ + -Ketoglutarate

Degradation of Amino Acids

NH3 -NH2 of -amino acids
Transamination Aminotransferase

-Ketoglutarate

NADH (NADPH)

Oxidative Deamination Glutamate Dehydrogenase

-ketoacids

-NH2 of glutamate

NAD+ (NADP+)

NH3 is Toxic Must be incorporated into UREA for disposal

Alanine Aminotransferase
Alanine -Ketoglutarate Alanine is deaminated to pyruvate Pyruvate is the -ketoacid of alanine

Pyruvate

Glutamate

Aspartate Aminotransferase
Oxaloacetate Glutamate Oxaloacetate is the -ketoacid of aspartate Formation of aspartate is favored during degradation Aspartate -Ketoglutarate Aspartate is required for the urea cycle

Protein Degradation Takeaway Points

Denatured in the stomach and degraded by specific proteases in the small intestine amino acids are absorbed Cellular proteins are continually synthesized and degraded (there is no storage form of protein). Degradation of amino acids proceeds through two steps Transamination (amino group is transferred to glutamate) Oxidative Deamination of Glutamate (generates free NH3 ) Ammonia is toxic and is detoxified in the liver Urea Cycle

Lehninger Figure18-10

The detoxification of ammonia occurs in the liver Glutamine is the predominant carrier of amino groups from most tissues Alanine transports amino groups from skeletal muscle and delivers its -ketoacid, pyruvate, to the liver. Glutamate NH3 and Aspartate

Urea Cycle

Lehninger Figure18-10

Occurs in both the mitochondria and cytosol Functions to convert ammonia to a non-toxic form Urea The two amino groups of urea are acquired from free ammonia (NH3) and aspartate The rate-limiting step Carbamoyl phosphate synthetase I

1. Ornithine transcarbamoylase 3. Argininosuccinase

Lehninger Figure18-10

2. Argininosuccinate synthetase 4. Arginase

The Urea Cycle and Citric Acid Cycle are Linked

Lehninger Figure 18-12

Oxaloacetate from the TCA cycle is the alpha-ketoacid of aspartate Fumarate is generated in the urea cycle and returns to the mitochondrial matrix for the TCA

Flow of Nitrogen from Amino Acids to Urea

Amino acids are processed transamination oxidative deamination Amino groups are collected in the form of free ammonia and aspartate Free ammonia and aspartate donate their amino groups to the formation of non-toxic urea

Urea Cycle Takeaway Points

Ammonia is sent to the liver to be removed from the body as urea The two amino groups of Urea are derived from free NH3 and Aspartate The urea cycle occurs in the mitochondria and cytosol of the liver The cycle is tightly linked to the citric acid cycle Urea is released by arginase and enters the blood stream Urea is filtered and excreted by the kidney.

Disposal of Amino Acids

The complete catabolism of amino acids (dietary or cellular) requires two reactions Transamination -Ketoacids Glutamate Oxidative Deamination of Glutamate -Ketoglutarate NH3

Amino Acid

NH3 + -Keto acid

Glucose or Acetyl CoA

Amino Acid Catabolism

The -ketoacids of amino acids are converted to Acetyl CoA and/or intermediates of the TCA Amino acids degraded to Acetyl CoA are ketogenic Amino acids degraded to intermediates of the TCA are glucogenic
Lehninger Figure-18-15

Lysine and Leucine are purely Ketogenic

Degradation of Phenylalanine

Lehninger Figure-18-23

Deficiency of Phenylalanine Hydroxylase or the cofactor tetrahydrobiopterin leads to Phenylketonuria

Phenylketonuria (PKU)
Inability to hydroxylate phenylalanine to tyrosine Primarily caused by deficiency of phenylalanine hydroxylase Phenylalanine is degraded by an alternate pathway phenylketones Phenylketones in urine musty odor Symptoms MR, failure to walk or talk, seizures, hyperactivity Neonatal Diagnosis
Lehninger Figure-18-25

Treatment Dietary restriction Tyrosine becomes essential

In-born Errors of Amino Acid Catabolism

Amino Acid Catabolism Takeaway Points

Degradation of amino acids proceeds through two steps Transamination Oxidative Deamination of Glutamate NH3 (Urea Cycle) -keto acids (further degraded) Degradation of the carbon skeletons of amino acids ultimately lead to synthesis of glucose (glucogenic amino acids) ketone bodies (ketogenic amino acids) Lysine and leucine are entirely Ketogenic Deficiency of phenylalanine hydroxylase PKU

General Outline- 2nd hour Synthesis of Non-Essential Amino Acids Amino Acids as Substrates Water-Soluble Vitamins Fat Soluble Vitamins

Biosynthesis of Nonessential Amino Acids

Essential Amino Acids
Can not be synthesized in the human body. Must be acquired through the diet PVT TIM HALL (Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Lysine and Leucine; Histidine and Arginine are required for growth)

Nonessential Amino Acids

Can be synthesized in the human body. Substrates Intermediary metabolites (oxaloacetate aspartate) Essential amino acids (phenylalanine tyrosine) (methionine cysteine) Tyrosine and cysteine are conditionally non-essential

Amino Acid Biosynthesis

Synthesized from intermediates of glycolysis, the citric acid cycle, and the HMP shunt Amino acids may serve as the precursor of other amino acids e.g. Serine glycine or cysteine Some amino acids are formed by the transamination of their -ketoacids
Lehninger Figure 22-9

pyruvate alanine oxaloacetate aspartate -ketoglutarate glutamate

Note: This figure includes essential amino acids that can not be synthesized by the human body

Synthesis of Amino Acids

NH3 -Ketoglutarate NADH (NADPH)

-NH2 of -amino acids

Transamination

Oxidative Deamination Glutamate Dehydrogenase

Aminotransferase

-ketoacids

-NH2 of glutamate

NAD+ (NADP+)

Transamination and oxidative deamination are readily reversible, according to the cells needs.

Amino Acids as Substrates

Amino acids are important precursors for the synthesis of specialized compounds in the human body Glycine is a precursor of the porphyrin ring of Heme oxygen binding in Hb substrate for bile pigments Several amino acids cooperate to synthesize compounds Creatine (an energy buffer in skeletal muscle) Glutathione (a critical reducing agent in RBCs) Nucleotide bases Several amino acids are decarboxylated to form special products Tryptophan Serotonin, Melatonin Tyrosine Dopamine, Norepinephrine, Epinephrine, Melanin Glutamate GABA Histidine Histamine

Synthesis of Catecholamines from Tyrosine

Tyrosine hydroxylase catalyzes the first step. Dopa is the substrate for the neurotransmitters Dopamine Norepinephrine Epinephrine
Lehninger Figure 22-29

The aromatic amino acid decarboxylase requires pyridoxal phosphate as a cofactor

Tyrosine is also the precursor of Melanin Key enzyme: Tyrosinase

Synthesis of Serotonin from Tryptophan

Tryptophan hydroxylase catalyzes the first step. The aromatic amino acid decarboxylase requires pyridoxal phosphate as a cofactor Pyridoxal phosphate is a cofactor in transamination reactions and decarboxylation reactions Serotonin functions in pain perception affective disorders regulation of sleep, temperature and blood pressure

Lehninger Figure 22-29

Decarboxylation of Amino Acids

Lehninger Figure 22-29

Histamine GABA Inhibitory Neurotransmitter Deficiency leads to seizures Potent Vasodilator Released from mast cells during allergic response Production in the stomach stimulates acid production

Amino Acids as Substrates Takeaway Points

Amino acids function as the precursors of other specialized compounds bile pigments nucleotides glutathione Specific Amino Acids and Their Products
Tyrosine Dopamine, Norepinephrine, Epinephrine, Melanin Tryptophan Serotonin, Melatonin Histidine Histamine Glutamate GABA

VITAMINS

Water-soluble

Fat-soluble
Vitamin A Vitamin D Vitamin E Vitamin K

NonB-complex
Ascorbic acid (Vitamin C)

B-complex

Energy-releasing
Thiamine (B1) Riboflavin (B2) Niacin (B3) Biotin Pantothenic acid

Hematopoeitic
Folic Acid Vitamin B12

Other
Pyridoxine (B6) Pyridoxal Pyridoxamine

Ascorbic Acid (Vitamin C)

Water-soluble vitamin Functions as an anti-oxidant Essential coenzyme in the hydroxylation of proline and lysine residues in collagen Required for normal connective tissue development and wound healing Vitamin C deficiency leads to Scurvy Sx sore, spongy gums loose teeth fragile blood vessels swollen joints anemia

Thiamine (Vitamin B1)

Functions as Thiamine Pyrophosphate (TPP) Coenzyme in oxidative decarboxylations pyruvate dehydrogenase -ketoglutarate dehydrogenase
Thiamine Deficiency Beriberi (B1B1) Common in diets consisting mainly of polished rice Sx tachycardia, vomiting convulsions, death Wernicke-Korsakoff Common in alcoholics Sx apathy, loss of memory rhythmic side-to-side movement of eyes

Riboflavin (Vitamin B2)

Precursor of the cofactors FMN and FAD Critical for oxidation-reduction reactions Riboflavin Deficiency Symptoms include dermatitis, cheilosis and glossitis

Niacin (Vitamin B3)

Precursor of the cofactors NAD+ and NADP+ Critical for oxidation-reduction reactions Tryptophan can compensate for niacin deficiency Niacin Deficiency = Pellagra Three Ds Dermatitis Diarrhea Dementia

Biotin
Coenzyme in carboxylation reactions Required for pyruvate carboxylase acetyl CoA carboxylase Biotin Deficiency is Rare Pantothenic Acid Component of coenzyme A Required for TCA, fatty acid synthesis and degradation Pantothenate Deficiency is Poorly Characterized

Folate
Found in leafy green vegetables Functions in one-carbon transfers as coenzyme tetrahydrofolate Important in the synthesis of amino acids, purines and thymine

Folic Acid Deficiency

Megaloblastic Anemia Failure to synthesize purine and thymidine nucleotides Accumulation of large, immature RBC precursors in the bone marrow Neural tube defects Spina bifida- failure to close the caudal portion Anencephaly- failure to close the rostral portion

Vitamin B12 (Cobalamin)

Required for the synthesis of methionine and degradation of odd-numbered fatty acids Only synthesized by microorganisms Obtained from eating foods derived from animals Absorption B12 binds to intrinsic factor, produced by the parietal cells of the stomach. Intrinsic factor and B12 are absorbed in the ileum Most B12 deficiency is due to autoimmune destruction of the parietal cells Pernicious Anemia Megaloblastic anemia CNS defects (irreversible)

Pyridoxine (Vitamin B6)

May be referred to as pyridoxine, pyridoxal, or pyridoxamine Converted to the biologically active form pyridoxal phosphate Is a cofactor for a variety of enzymes, especially those associated with amino acid catabolism

Reaction Type Transamination Deamination Decarboxylation

Example Oxaloacetate + Glutamate Aspartate + -ketoglutarate Serine Pyruvate + NH3 Histidine Histamine + CO2

Vitamin
Folic Acid Vitamin B12 Vitamin C Vitamin B6 Vitamin B1 Niacin Riboflavin Biotin Pantothenic Acid

Function
Transfer of 1-carbon units Synthesis of purines and thymine Degradation of oddnumbered fatty acids Antioxidant Cofactor for Hydroxylations Cofactor for a.a. catabolism Cofactor for oxidative decarboxylation reactions Electron Transfer Electron Transfer Carboxylation reactions Acyl carrier

Deficiency
Megaloblastic Anemia Neural Tube Defects Megaloblastic Anemia Neuropsychiatric Sx Scurvy (loose teeth & Poor wound healing) Rare Beriberi Wernicke-Korsakoff Pellagra (3 Ds) Rare Rare Rare

Fat Soluble Vitamins ADEK

Absorbed from the diet in mixed micelles Pancreatic insufficiency can lead to steatorrhea and deficiency of fat-soluble vitamins (ADEK) Function much like steroid hormones readily diffuse through the lipid membrane have nuclear receptors alter gene expression Are stored in fat deposits in the human body. Excessive intake can lead to toxicity.

Vitamin A (Retinol)
Retinoids class of compounds related to Vitamin A required for vision, growth and epithelial tissues Deficiency causes night blindness and xerophthalmia Excess Vitamin A leads to toxicity teratogenic

Vitamin E
Primarily functions as an antioxidant, preventing the nonenzymatic oxidation of compounds by molecular oxygen or free radicals Deficiency in adults is rare Least toxic of the fat-soluble vitamins (ADEK)

Vitamin D
Two precursors Ergocalciferol (Acquired through the diet) 7-Dehydrocholesterol (Synthesized in the skin) Precursors are activated by hydroxylation position 25- in the liver position 1- in the kidney Active 1,25 dihydroxycholecalciferol Synthesis of active Vitamin D is stimulated by parathyroid hormone in response to low blood calcium.

Lehninger Figure-10-20

Vitamin D (contd)
Vitamin D functions to maintain blood calcium levels increases calcium absorption in the intestine limiting loss of calcium through the kidney promoting bone resorption in conjunction with PTH Vitamin D Deficiency Rickets (in children) Collagen matrix of bone is formed but is not completely mineralized Soft, pliable bones Osteomalacia (in adults) Existing bones are demineralized and are much more susceptible to fracture Vitamin D deficiency can be a result of insufficient UV exposure dietary deficiency advanced renal disease

Vitamin K
Vitamin K is essential for post-translational modification of clotting factors II, VII, IX, and X Blood clotting factors are synthesized in the liver where they undergo Vitamin K-dependent carboxylation of their glutamate residues. Glutamate carboxylation is required for clotting factors to interact with activated platelets at the site of a wound. Modified glutamate has an overall charge of -2. Warfarin is a competitive inhibitor used as an anticoagulant in patients at risk of heart attack or stroke. Deficiency of Vitamin K is rare in adults microbes in gut synthesize Vitamin K

Blood Clotting Cascade

Damage to the endothelium exposure of subendothelial cells Von Willebrand Factor and collagen recruit & activate platelets 1- expression of fibrinogen receptors 2- expression of phosphatidylserine phosphatidylserine binds Ca2+ positively charged calcium recruits the negatively charged clotting factors Intrinsic Pathway all the components required for clot formation are intrinsic to the blood Extrinsic Pathway- requires cooperation of tissue factor at the site of the wound for clot formation

Blood Clotting Cascade (contd)

Intrinsic Pathway XI Exposed negative Extrinsic Pathway
surface

X
VII
Cells expose Tissue Factor

XIIa IXa
+

XII

XIa

VIIa

IX

VIIIa

Fibrinogen Thrombin Fibrin

Prothrombin

Xa + Va

Factors II, VII, IX and X are Vitamin K dependent Factors V and VIII are coenzymes

Fat-Soluble Vitamins ADEK Vitamin

Vitamin A

Function
Reproduction Vision Maintenance of epithelial tissue Calcium Uptake Antioxidant Cofactor for -Carboxylation of glutamate in clotting factors

Deficiency
Impotence Night Blindness Xerophthalmia Rickets (children) Osteomalacia (adults) Rare RareCompetitively inhibited by Warfarin

Vitamin D Vitamin E Vitamin K