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04
7, June, 2015 Dr. Menorca | Cofactors, Coenzymes and Prosthetic Groups

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OUTLINE
I. Cofactors, Conezymes and Prosthetic Groups
II. Reactions
a. Redox Reaction and Group Transfer Reaction
b. Isomerization reaction & reactions that form
covalent bond
III. Coezymes for Redox Reaction
a. NAD & NADP
b. FMN & FAD
c. Coenzyme Q
IV. Coezymes for Group Transfer Reaction
a. Coenzyme A & TPP
b. PP & Biocytin
c. Tetrahydrofolate & cobalamine

COFACTORS, COENZYMES AND PROSTHETIC GROUPS Zinc Proteases:


a. Carboxypeptidase
DEFINITION OF TERMS b. Thermolysin
COFACTORS o With identical active sites
o Same mechanisms as in CARBONIC
 Small organic or inorganic molecules that an ANHYDRASE
apoenzyme requires for its activity. o Additionally, they stabilize the TRANSITION
 Generalized term STATE
 They are coenzymes and metal ions
respectively. Metals that Form Chelate without Acid Catalysis:
- Coenzymes a. Mg in Creatine Kinase
o Ex. Vitamins (B vitamins) o Serves to neutralize the negative charge density
o Organic cofactors of ATP and facilitate binding to enzyme.
- Metal ions o Ternary complexes of this conformation are
o Inorganic cofactors known as “substrate-bridged” complexes (Enz–
o Required in approximately 2/3 of all S–M). eg:
enzymes; can participate in reactions by: CK-Adenine-Ribose-(PO3)3
 Chelate formation | |
 Acting as Lewis Acid
o Examples: Zn in Carbonic Anhydrase: Creatine Mg

b. Mg in Pyruvate Kinase
o Mg chelates ATP to enzyme.
 Mg, no ATP binding to enzyme.
o “Metal-bridged” (Enz–M–S).

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PK-Mg-Adenine-Ribose-(PO3)3 it from the apocytochrome. This is an example of an


inorganic cofactor acting as a prosthetic group.
|
In the enzyme lysine oxidase, the copper is
Pyruvate loosely bound but is required for the enzymes to be
active. This is an example of an inorganic cofactor that
METAL COMPLEXES IN REDOX: is not a prosthetic group.
The biocytin is tightly bound to the enzyme
a. Nonheme iron proteins (Fe-S proteins)
acetyl CoA carboxylase without which, carboxylation
1) Sulfur-bridged iron chelates.
cannot proceed. This is an example of a coenzyme
2) Have reasonably low redox potential (Eo’)
(organic cofactor) that is a prosthetic group.
3) Electron transfer reaction. Most coenzymes dissociate from the
b. Heme iron proteins (CYTOCHROMES) apoenzyme after the completion of the chemical reaction
1) Apoprotein is coordinated to heme. and therefore, they are evidently loosely bound to the
2) Iron undergoes reversible 1e- transfer apoenzyme and therefore not a prosthetic group.
3) Fe serves struc’ral & chm’cal role.
c. Metal activators of O2 (Cu and Fe)
1) Dopamine B-hydroxylase (Cu) REACTIONS
2) Cytochrome P450 (heme Fe)  Enzyme Classes (IUB) 1, 2, 5, 6
 Hydrolases, lyases and enzymes of digestion
COENZYMES reactions do not require coenzymes because of
the lytic nature of their reactions
 Small organic molecules that function with the
enzyme in the catalytic process OXIDATION-REDUCTION REACTIONS
 Often derivatives of vitamins (usually B Coenzymes can act as intermediate carriers of
vitamins) electrons in RedOx reactions.
 Often, has an affinity for the enzymes much
similar to that of the substrate, thus it is
considered to be a second substrate.
 Sometimes, it is covalently bound to the
apoenzymesand functions at or near the
active site in catalysis.
 In others, it is midway between the two
extremes.

PROSTHETIC GROUP eg, Oxidation of ethanol to acetaldehyde


Enzyme: alcohol dehydrogenase
 Cofactors that are tightly bound to the Coenzyme:NAD+ [oxidizing agent; acts as carrier of
apoenzymes hydride ion (:H-)]
 Some do not detach from the enzyme after the GROUP TRANSFER REACTION
chemical reaction
Examples: Functional group (G) is transferred from donor
In cytochromes, the heme prosthetic group is molecule (D) to an acceptor molecule (A).
very tightly bound and requires strong acids to dissociate

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D–G + A A–G + D Conversion of 2-phosphoglycerate to 3-


phosphoglycerate
D – G + IC  IC – G + A –D  A – G + IC Enzyme: mutase (phosphoglyceromutase)
Action: transfer of phosphate group from carbon 2 to
Coenzyme can either be the ultimate acceptor (A, carbon 3
eg, dehydration reaction) or the group carrier (IC, Coenzyme: Cobamide
eg, transamination reactions).

Conversion of glucose-6-phosphate to fructose-6-


phosphate
*glucose and fructose are isomers (same chemical
structure but different conformations  isomerization
reaction)
Enzyme: isomerase (phosphoglucoisomerase)
Action: aldose-ketose isomerization
Enzyme: Aspartate Transaminase Coenzyme: Cobamide
D-G: Aspartic Acid
A-G: Glutamic Acid
IC: Pyridoxal PO4 (carrier of -NH2 group)

ISOMERIZATION REACTIONS

 transfer of groups within molecules to yield


isomeric forms
 requires a cofactor
 3 types:
 cis-trans and aldose ketose REACTIONS THAT FORM COVALENT BONDS
interconversion
 epimerases and racemases– inversion of Fatty Acid Synthesis
asymmetric carbon atoms Enzyme: Ligase (Fatty Acid Synthetase)
epimerase – interconverts epimers Synthetase – catalyze the formation of a new bond
(compounds having the same chemical between substrates with the participation of ATP
formula but differ in the spatial arrangement Coenzyme: Acetyl-coA
around a single carbon atom) Action: Elongation of a fatty acid
racemase – conversion of D to L isomer or A FA is elongated by 2 C due to the formation of a
covalent bond between the terminal C (carboxyl C) of the
vice-versa
original fatty acid and the methyl C of the coenzyme
 mutases - intramolecular group transfer; acetyl-coA.
conversion of one structural isomer to
another

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Both function as:


1. Intermediate in transfer of 2 electrons (e-) between e-
donor and acceptor.
2. A common pool of e- that arise from may oxidative
reactions and can be used for various reductive
reactions.
ADENINE, RIBOSE, and PYROPHOSPHATE
COENZYMES FOR REDOX REACTIONS components of NAD are involved in the binding of NAD to
the enzyme.
NAD & NADP - 2’ pyrophosphate of NADP prevents binding of
NADP to dehydrogenases with aspartate
NIACIN - pyridine 3-carboxylic acid residue in the binding site (as in dehydrogenase
specific only for NAD) because of the charge
interaction.

It is converted to 2 coenzymes involved in REDOX


a. NAD (Nicotinamide Adenine Dinucleotide)
b. NADP (Nicotinamide Adenine Dinucleotide Phosphate)

*NAD+& NADP+ represent the OXIDIZED form


NADH & NADPH represent REDUCED form

Some dehydrogenases are specific for NAD, and others


for NADP, while others functions with either.
- This allows for specificity and control over
dehydrogenases that reside in the same
subcellular compartment. NAD Structure
ANALYSIS OF STRUCTURE:
NAD+-NADH pair – act primarily in a catabolic direction; 1. C4 has a LOW electron density.
in other words nicotinamide nucleotide-linked enzymes 2. –CONH2 (functional group on the right of C4)
that oxidize substrates (dehydrogenases) usually use attracts e- to itself due to HIGH electronegativity
NAD+. scale of Oxygen.
3. N+ attracts electron to itself as it is electrophilic.
NADP+-NADPH pair – act primarily in anabolic pathways;
nicotinamide nucleotide-linked enzymes that reduce *No.2 and 3 renders C4 low in e- density
substrates (reductases) usually use NADPH.

An exception is two dehydrogenases in the pentose


phosphate pathway, which convert NADP+ to NADPHand
represent the principal route for synthesis of the reduced
nucleotide.

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Example 1 Oxidation of alcohol to aldehyde

Reaction Oxidation

Dehydrogenase (Alcohol
Enzyme
Some Reactions Participated in by NAD and NADP dehydrogenase)

NAD+ (acceptor of e- and H+); The


Electron Transport Chain (ETC) in Coenzyme
coenzyme was reduced to NADH
Example 2 the
mitochondria
Oxidative Phase of Pentose
Phosphate
Example 3
Pathway (Aka: Hexose
Monophosphate Shunt)

Reaction Redox
Enzyme NADH Dehydrogenase (Complex I) Reaction Oxidation
NAD+; NADH is the first source of
Glucose-6-phosphate
Coenzyme electron that will be passed down the
Enzyme dehydrogenase and 6-
chain.
phosphogluconate dehydrogenase

Coenzyme NADP+

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FMN & FAD

 Both function in oxidation-reduction reactions by


accepting and donating two electrons in the
isoalloxazine ring
 Can be electron acceptor (accepts 1 electron)
and form flavinsemiquinone (a free radical)

 Tend to be bound much tighter to their


apoenzymes than the niacin coenzymes and
often function as prosthetic group
 The most important in its function is the
isoalloxazine ring

Some of the enzymes that utilize these


coenzymes include dehydrogenases,
oxidases, reductases, and oxygenases

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COENZYME Q ( UBIQUINONE; UBIQUINOL) Structure- Function Relationship

It is a lipid soluble benzoquinone with a long isoprenoid


tail

Also called ubiquinone, because it is ubiquitous


(abundant) in biologic systems

- Can be synthesized in the body, not derived from


vitamin
- Plays a central role in coupling electron flow to
proton movement since it carries both electrons
and protons
- Has a long hydrophobic side chain consisting of
6-10 isoprene units, depending on the source of
CoQ
- Freely diffusible (small and hydrophobic) within
the lipid bilayer of the inner mitochondrial
membrane
- Ubiquinone can accept one electron to become
the semiquinone radical (QH) or two electrons
to form ubiquinol (QH2)
- The quinone portion – oxidized and reduced by
addition of 2 reducing equivalents (2H+ and 2e-)
- Like the flavoprotein carriers, it can act at the
junction between a two-electron donor and a
one-electron acceptor
- CoQ can accept hydrogen atoms both from
FMNH2, produced by NADH dehydrogenase, and
from FADH2 (Complex II), which is produced by
succinate dehydrogenase and acyl CoA
dehydrogenase
- Can act to transfer electrons from Complex I and
II to Complex III

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COENZYMES FOR GROUP TRANSFER REACTION

COENZYME A & TPP

COENZYME A (CoA, CoASH, or HSCoA)


- Derived from panthotenic acid
- Acts by group transfer reaction specifically acyl
group transfer
- In CoASH, the H in the thiol of CoASH (the H in
CoAS—H) is acidic due to orbital resonance in sulfur
which is a nucleophile, this acidity is responsible for Reactions that involve Coenzyme A
the coenzyme activity 1. Biological esterification (esterases)
- This is proven by the inactivity of both CoAS—R
(where R can be any group except H) and CoA-S—
S-CoA
- The thiol can react with carboxylic acids forming
thioesters, this makes them functional in acyl group
transfers

2. Fatty acid synthesis [F.A. synthase (not synthetase, mali


yung nasa picture)]

(example of thioester formation between CoA and acetic 3. Others


acid which is a carboxylic acid) a) Cholesterol biosynthesis
b) Glycoprotein biosynthesis
STRUCTURE FUNCTION RELATION c) Gene expression
d) Ketone bodies formation
CoA = Cysteine + Pantothenic Acid (vitamin B5) + adenosine e) N-acylation of hormones
triphosphate (ATP) f) Heme formation
g) Neurotransmitter synthesis
h) Many others  (WTF?!!)

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THIAMIN (B1)

 The vitamin that is rapidly converted to the


coenzyme thiamine pyrophosphate (TPP)
 Required for the key reactions catalyzed by
pyruvate dehydrogenase complex and alpha-
ketoglutarate dehydrogenase complex
 Cellular energy generation is severely
compromised in thiamine deficiency
THIAMIN PYROPHOSPHATE (TPP)

- One of the three (3) catalytic cofactors in pyruvate


dehydrogenase complex: Pyruvate dehydrogenase
(TPP), Dihydrolipoyl transacetylase (lipoamide), and
Dihydrolipoyl dehydrogenase (FAD)
- It transfers aldehyde and ketone groups
(transketolation reaction)

STRUCTURE FUNCTION RELATION

- PP & BIOCYTIN
-
-
- Thiazole ring is the one that is directly involved in the VITAMIN B6
transfer of ketone (or aldehyde) and decaroboxylation - Efficiently converted by the body to pyridoxal
of alpha keto acids phosphate, which is required for the synthesis,
- Carbon #2 (encircled with blue) is the main site of TPP, catabolism and interconversion of amino acids
it is CARBANIONIC due to the resonance inductive - Most versatile enzyme cofactor
effect of pyrimidine and nulceophilicity of sulfur - Vitamin B6s are readily soluble in water,
- Stable in acid, unstable in alkali
Reactions that involve TPP
- Easily destroyed with UV light (ie. sunlight)
1. Decarboxylation (Decarboxylase) - well-absorbed through the mucosa of the small
intestine

Forms of
Structure
Vitamin B6

2. Transketolation (Transketolase) Pyridoxine

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Pyridoxamine

The Reaction:
Pyridoxal

Formation of Pyridoxal Phosphate via Phosphorylation

PYRIDOXAL PHOSPHATE (PP OR PLP)


- Active site of the resting aminotransferase
Note: Many pyridoxal phosphate-requiring reactions
contain this enzyme covalently attached to an involve transamination, but the ability of the Schiff base to
epsilon amino group of lysine residue that forms transfer electrons between different atoms allows this
part of the primary structure of the transferase coenzyme to participate in the removal of other groups as
- The coenzyme attachment, --CH=N--, is called a in:
Schiff base, its carbon from the aldehyde group
1) Decarboxylation of amino acids
of coenzyme and its nitrogen from the lysine
2) Dehydrative deamination of serine and threonine
residue of the enzyme
3) Desulfurative deamination of cysteine
- The amino group of substrate amino acid
displaces the lysine epsilon amino group and a The biochemical role of PP is associated with more than
Schiff base forms between the substrate and the 60 enzymes involved in:
coenzyme
1) Synthesis of neurotransmitters
- The new Schiff base takes the form of an
2) Synthesis of sphingolipids
aldimine and ketimine in tautomeric equilibrium
3) Amino acid metabolism
- Hydrolysis of the ketamine liberates an alpha
4) Synthesis of heme precursors
keto acid with the amine group left to the
5) Component of glycogen phosphorylase
coenzyme that became pyridoxamine
6) Cofactor in the biosynthesis of niacin from
- The reverse process is the reaction of an alpha
tryptophan
keto acid to the amine group resulting in the shift
7) Biosynthesis of Coenzyme A
of the double bond of the Schiff base which when
8) Production of ethanolamine and taurine
hydrolyzed, a new amino acid is liberated
9) Cofactor for lysine oxidase which is involved in
- Subsequently, pyridoxal phosphate reforms its
the biosynthesis of elastin and collagen
Schiff base with the resting enzyme

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1.04 Dr. ABC XYZ | Topic

BIOCYTIN

Biotin  1 CARBON fragment is carried by THF as:


a. N5N10CH2FH4 or 5,10 methylene THF

Biocytin

b. N5N10CHFH4 or 5,10 methenyl THF

- Precursor: Biotin
- Transfer: CO2
- Occurs naturally in blood serum and urine
- The enzyme methyl malonyl CoA carboxylase
utilize this as cofactor
- It is a covalently bound prosthetic group of the c. N10C=O FH4 or N10 formyl THF
enzymes:
o pyruvate carboxylase
o acetyl CoA carboxylase
o propionyl CoA carboxylase
- End product of the proteolytic degradation of
biotin dependent carboxylases, e. g. pyruvate
carboxylase. d. N5CH= NHFH4 or N5 formimino THF
- To recycle the vitamin biotin, biocytin has to be
cleaved by the enzyme biotinidase.
- Used as neuronal tracer in brain research
(histological stain for nerve cells)

TETRAHYDROFOLATE & COBALAMINE REACTIONS:

TETRAHYDROFOLATE (THF; H4 FOLATE) 1.



 Coenzyme derived from folic acid (reduced form, 2.
one of the B vit.)
 Various 1C THF are used in biosynthetic rxns 3. De novo
such as synthesis of choline, serine, glycine, biosynthesis of purine
purines and dTMP(Deoxythymidine rings- synthesis of
nucleotides from
monophosphate)
simple materials like
 Abbreviated formula: amino acids

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1.04 Dr. ABC XYZ | Topic

VITAMIN B12 (COBALAMINE OR EXTRINSIC FACTOR)

 Consists of cobalt in coordination state of six: in four


positions by corrin ring, fifth by benzimidazole
nitrogen and in sixth by a ligand (X).

4. Conversion of UMP to TMP (Pyrimidine de novo


biosynthesis)

 Acid hydrolysis in stomach or trypsin digestion in


the intestine carries it to the ileum for absorption
 Megaloblastic anemia -associated with B12
deficiency. There is accumulation of N 5 methyl
THF and deficiency in THF derivatives needed
for purine and dTMP (TMP) biosynthesis.
Inhibition to convert N5-methyl THF to THF
*Phenylalanine hydroxylase is Tetrahydrobiopterine- affects the ability of the cell to convert it to
dependent. This coenzyme is related to folic acid bec. of polyglutaminated form which is biologically its
its Pteridine group but it is not a vitamin. It is most potent form.
synthesized from GTP.
*Pteridine group- heterocyclic compounds composed of
pyrimidine and pyrazine rings.

Dihydrobiopterin (BPH2) produced during oxidation of


aromatic AA and then reduced to
TETRAHYDROBIOPTERINE (BPH4).

Synthesis of BPH4:

*Catecholamine synthesis (starts with Tyr


hydroxylase) is affected by BIOPTERINE
DEFICIENCY or dihydrobiopterine reductase.

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