BIOCHEMISTRY 09/03/2019

Coenzymes Shifting 1

Trans 4
Dr. Menorca
[SUBJECT]
OUTLINE  Enzyme which accepts hydroxyl group from water
 Zn proteases
I. Definition of Terms a. NAD/NADP MM/DD/YYYY  Carboxypeptidase and thermolysin
II. Inorganic Cofactors b. FMN/FAD Shifting #  With identical active sites
A. Metals that form c. Coenzyme Trans #  Chelate forming, acting as lewis acid
chelate without acid Q  Stabilize transition state
catalysts C. Coenzymes for Group
B. Metal [SUBJECT]
complexes in Transfer Reactions
redox a. Coenzyme A
III. Organic Cofactors b. TPP (Martin)
A. Reactions requiring c. PP
[SUBJECT]
coenzymes d. Biotin
B. Coenzymes for Redox e. THF
Reactions f. Cobamide
I. DEFINITION OF TERMS

Cofactors
 Small inorganic/organic molecule needed for the activity of the
apoenzyme
 APOENZYME
 Protein part of the holoenzyme
 HOLOENZYME
 Apoenzyme + cofactor Figure 1. Reaction Mechanism of Carbon Anhydrase
 Activated form of enzyme
 Two types of cofactor A. METALS THAT CHELATE WITHOUT ACID
 Inorganic cofactors CATALYSTS
 Metal ions (Metal-activated enzyme)
 Mg2+ in creatine kinase
 Prosthetic group of a particular enzyme
 Neutralize the negative charge density of ATP and facilitate
 Organic cofactors
binding to the enzyme
 Coenzymes
 Ternary complexes of this conformation are known as
 Derived from vitamins
Substrate-bridges are formed (Enz – Substrate – Metal)
 Second substrate due to its affinity to enzyme
 Prosthetic group of particular enzyme  Mg in pyruvate kinase
 Associates with the enzyme either directly or by forming  Mg chelates ATP to enzyme
cofactor substrate complex  Without Mg, no ATP will bind to the enzyme
Prosthetic Groups  “Metal-bridge” (Enz-M-S)
 Cofactors tightly bound to the apoenzyme
 Can be organic/inorganic
 Incorporated to the protein structure via covalent and non-
covalent forces
 Metal ions are the most common type
 Ex. Pyridoxal phosphate (PLP), Flavin
mononucleotide(FMN), thiamin pyrophosphate (TPP)
Coenzymes
 Recyclable shuttles
 Transports substrate within the cell
 Stabilize species that are too reactive to persist in the presence
of water or organic molecules that permeates the cell
 Serves as an adapter or handle that facilitates the recognition
Figure 2. Reaction Mechanism of Pyruvate Kinase
and binding of chemical groups by their target enzyme
 Ex. Acetate (coenzyme A), glucose (UDP), methyl groups
B. METAL COMPLEXES IN REDOX
(folate), oligosaccharides (dolichol)

II. INORGANIC COFACTORS  Non-heme iron proteins (Fe-S proteins)

 Comprises 2/3 of all enzyme - Sulfur-bridged iron chelates
- Have reasonably low redox potential (Eo’)
 Metalloenzyme
- Electron transfer reaction
 Comprises 1/3 of all enzyme
 Contains tightly bound Fe, Co, Cu, Mg, Mn, and Zn
 Heme iron proteins (CYTOCHROMES)
 Served as prosthetic groups - Apoprotein is coordinated to heme
 Metals participate in enzyme reaction: - Iron undergoes reversible 1e- transfer
 Chelate formation - Fe serves structural & chemical role
 Acting as Lewis acid (electron acceptor) or bases to render - Has a porphyrin ring structure
substrate more electrophilic (electron-poor) or nucleophilic
(electron-rich)
 Ex. Zn in carbonic anhydrase

BCHM Abihin, Angli, Apigo, Catudio, Junio, Leyso, Mago, Ong, Peconcillo, Sampaga 1 of 9
 Oxidizing agents NAD+ and NADP+
(undergone reduction)
Reducing agents NADPH and NADH
(undergone oxidation)

B. Group Transfer Reaction

 Functional group (G) is transferred from donor molecule (D) to

an acceptor molecule (A)

(Dehydration) D–G+A→ A–G+D

(Transamination) D – G + IC → IC – G + A –D → A – G + IC

 Coenzyme as
Ultimate acceptor: Dehydration reaction (A is the coenzyme)
Figure 3. Iron in heme of hemoglobin Intermediate carrier: Transamination reaction (IC)

 Metal activators of O2 (Cu and Fe)

- Dopamine B-hydroxylase (Cu)
- Cytochrome P450 (heme Fe)

III. ORGANIC COFACTORS

 COENZYMES are intermediate carriers of:
- Functional groups
- Specific atoms
- Electrons
*They are all transferred in the overall enzymatic reaction
 Reactions that require coenzymes:
- IUB 1, 2, 5 & 6
Class 1 – Oxidoreductase
Class 2 – Transferases
Class 5 – Isomerases
Class 6 – Ligases Figure 6. Transaminase reaction. The amine group (-NH2) from Asp is held on
 Reactions that do not require coenzymes: by the PLP (coenzyme). Afterwards, the amine group is transferred to the α-
ketoglutaric acid to produce Glu.
- IUB 3 AND 4
Class 3 – Hydrolase Enzyme Aspartate Transaminase
Class 4 – Lyases D-G Aspartic Acid
* Do not require coenzymes because of the lytic nature of A-G Glutamic Acid
their reactions IC PLP (carrier of -NH2 group)
A. REACTION REQUIRING COENZYMES
C. Isomerization Reaction
A. Oxidation-Reduction Reaction
 Coenzymes can acts as intermediate carriers of electron in  Transfer of groups within molecules to yield isomeric forms
RedOx reactions  3 types:
*Remember: Hydrides can carry 2e, hence gain of H- is gain of - Cis-trans and aldose-ketose interconversion
electrons=reduction - Epimerases and racemases– inversion of asymmetric
carbon atoms

1. Epimerase – interconverts epimers (compounds having

the same chemical formula but differ in the spatial
arrangement around a single carbon atom)
2. Racemase – conversion of D to L isomer or vice-versa
Figure 4. Alcohol dehydrogenase reaction using NAD as coenzyme 3. Mutases - intramolecular group

Substrate Ethyl alcohol

Product Acetaldehyde
Enzyme Alcohol dehydrogenase
Coenzyme NAD

Figure 5 . Conversion of NADP+ to NADPH (a reversible reaction)

BCHM Coenzymes 2 of 9
 Conversion of 3-phosphoglycerate to 2-phosphoglycerate  NAD+& NADP+ (oxidizing agents) represent the OXIDIZED form
 NADH & NADPH (reducing agents) represent REDUCED form -
derived from niacin
 Function as intermediate in transfer of 2 electrons between and
electron donor and acceptor
 Act as a common “pool” of electrons that arise from many
oxidative reactions and can be used for various reductive
reactions
 ADENINE, RIBOSE & PYROPHOSPHATE part of NAD binds to
the enzyme
 The -PO3 2- of NADP prevents binding to the enzyme with Asp
(COO-) due to charge-charge interaction
 NAD+ is not a prosthetic group so it disengages to acid and is in
amount in electron transport chain
NADPH: large amount in pentose phosphate pathway

Figure 7. Cofactor-dependent phosphoglyceromutase. The phosphorylated His

residue transfers the –PO42- group at C2 position making a 2,3-BPG intermediate.
Then the – PO42- group at C3 is removed to give 2PG.

mutase
Enzyme (cofactor-dependent
phosphoglyceromutase) Figure 9. Structure of NADH
transfer of phosphate group C4 has a low electron density because the amide group -CONH2
Action
from C2 to C3 (functional group at C4) attracts electron to itself due to the
Coenzyme phospho-histidine electronegativity of oxygen
-N+ attracts electron to itself because it is an electrophile
● Reactions that form covalent bond -The rest of the structure is used to stabilize and hold the small
- Example: Fatty acid synthesis reactive center.
- A fatty acid is elongated by 2C due to the formation of a
covalent bond between the terminal C (carboxyl C) of the original Some Reactions Participated by NAD AND NADP:
fatty acid and the methyl C of the acetyl-coA 1. Oxidation of alcohol to aldehyde:

Figure 10. Reaction Mechanism of Alcohol Dehydrogenase

Figure 8. FA synthesis. A FA is activated via ATP. The phosphorylated FA is 2. Oxidation of aldehyde to acid:
then converted into a thioester, acetyl-CoA via Coenzyme A.
Ligase
Enzyme
(Fatty Acid Synthetase**)
Action Coenzyme A (CoA)
Coenzyme Elongation of a fatty acid

**Synthetase – catalyze the formation of a new bond between

substrates with the participation of ATP

Table 1. Summary of Coenzymes for REDOX reactions

Coenzyme Vitamin Source Function Figure 11. Series of oxidation from a 1° Alcohol to Aldehyde to Carboxylic
Niacin Acid
NAD
(pyridine 3-
NADP 3. Electron Transport Chain of Mitochondria:
carboxylic acid) All are for Electron
FMN (Flavin Transfer NAD+ is the coenzyme of some of the mitochondrial
Riboflavin dehydrogenase.
Mononucleotide)
Coenzyme Q Ubiquinone

B. COENZYME FOR REDOX REACTIONS

NAD+/NADP+
 Nucleotide Adenine Dinucleotide (NAD+)
 Nucleotide Adenine Dinucleotide Phosphate (NADP+)

BCHM Coenzymes 3 of 9
 Figure 12. Reaction Mechanism of Lactate Dehydrogenase
4. Oxidative Phase of Pentose Phosphate Pathway
Coenzyme Q (Ubiquinone, Semiquinone, Ubiquinol)
 The terminal electron acceptor of Complex I.
 Can act as one or two electron acceptor due to the presence of
a stable semiquinone intermediate.
 Has long hydrophobic side chain consisting of 10 isoprene
units, which is buried in the membrane lipid bi-layer.
 Freely diffusible in the membrane and can act to transfer
electron from complex 1 and complex 2 to complex 3.
 Derived from ubiquinone.

Figure 13. Pentose Phosphate Pathway

FAD+/FMN
 Flavin Adenine Mononucleotide
 Flavin Adenine Dinucleotide
 Both function in Redox reaction by accepting and donating 2
electron in the isoalloxazine ring.
 Tend to be bound much tighter to their apoenzyme than the
niacin coenzymes (Prosthetic groups).
 Derived from Riboflavin

Figure 15. Structures of Ubiquinone, Semiquinone and Ubiquinol

C. COENZYMES FOR GROUP TRANSFER REACTIONS

Table 2. Vitamin Sources and Functions of Coenzymes

Coenzyme Vitamin Source Function
Coenzyme A Pantothenic A Acyl group
transfer
TPP Thiamine Aldehyde
transfer
PP Pyridoxine Amino group
Figure 14. Structure of FAD+ and FMN transfer
Biocytin Biotin CO2 transfer
 Some of the enzymes that utilizes this include: THF Folic Acid One-C group
1. DEHYDROGENASES (FAD FADH2) transfer
2. OXIDASES (FADH2 O2 H2O) Cobamide Vitamin B12 Alkyl group
3. REDUCTASES (FADH2 H2) transfer and
4. OXYGENASES (FADH2 H2O2) isomerization

Coenzyme A (CoA, CoASH, or HSCoA)

 Derived from pantothenic acid (Vitamin B5)
 CoA = Cys + Vit. B5 + ATP
 for acyl group transfer reaction
 coenzyme a has an acidic hydrogen (CoAS---H) because of
orbital resonance in S which is a nucleophile
 CoAS-R and CoAS-SCoA are both inactive pointing to the
SH group to be responsible to the coenzymic activity.
 More than 70 enzymes utilize this coenzyme and its
derivatives

BCHM Coenzymes 4 of 9
 Figure 16. Pantothenic acid and coenzyme A. Asterisk
shows site of acylation by fatty acids.

 Reactions:
1. Biological Esterification

Figure 17. Cholesterol (Mevalonate) Biosynthesis

b. Glycoprotein Biosynthesis

2. Fatty Acid Synthesis

a. For elongation of fatty acid chain

3. Others
a. Cholesterol Biosynthesis

Figure 18. Glycoprotein Biosynthesis

c. Gene Expression
i. Histone covalent modification is a
determinant of gene activity.
ii. Modifications are dynamic and
reversible
iii. Histone acetylation and
deacetylation.
iv. Histone acetylase require Coenzyme

BCHM Coenzymes 5 of 9
 d. Ketone Bodies Formation

Figure 23. Synthesis of Neurotransmitters

h. Many Others

TPP (Thiamin Pyrophosphate)

 The thiazole ring is directly involved in the transfer of
aldehyde/ketol & in decarboxylation of alpha keto acid

Figure 24. Thiamin, thiamin diphosphate, and the carbanion form.

 Carbon#2(*) is carbanionic.
 this coenzyme from vit B1(thiamine) is required for the reaction
catalyzed by pyruvate DH complex composed of: [pyruvate
dehydrogenase (TPP); dihydrolipoyl transacetylase (lipoamide);
and dihydrolipoyl dehydrogenase (FAD)].
 aldehyde transfer and ketol group transfer (transketolation
reaction)
 cellular energy generation is severely compromised in thiamine
deficiency

Figure 19. Formation of Ketone Bodies

e. N-acylation of Hormones

Figure 25. Reactions catalyzed by pyruvate DH complex: pyruvate

dehydrogenase

Figure 20. N-acylation of Hormones  Transketolation reaction

 aldehyde transfer and ketol group transfer
f. Heme Formation
 in the ketone groups, the carbon (C=O) is positive due to the
i. Phorphobilinogen - 1st precursor of
pyrole electronegativity of oxygen and cannot bind to another ketone
because they will repel each other
 Transketolase enzyme converts 1 ketone substrate to make the
carbon negatively charged and bond with another ketone
group’s carbon

Figure 26. Transketolation

PP (Pyridoxal Phosphate)
Figure 21. Heme formation

g. Neurotransmitter Synthesis
BCHM Coenzymes 6 of 9
 2. Synthesis of Sphingolipids

Figure 27. Structure of Pyridoxal Phosphate

 At the green circle, it can be change into *CH2OH; *CH2NH2;

*CHO
 VITAMIN B6 (PYRIDOXINE, PYRIDOXAMINE, PYRIDOXAL)
 Found in the active site of “resting amino transferases” attached
to an epsilon amino group of a lysine residue
 It is the coenzyme for transamination (aminotransferases) and
specific decarboxylation (decarboxylases)
 Pyridoxine is converted by the body to pyridoxal phosphate
(using ATP) which is required for the synthesis, catabolism and
 interconversion of amino acids. Figure 30. Synthesis of Ketosphingolipids
 Schiff base is formed through the condensation of an amine
with a carbonyl group. 3. Amino Acid Metabolism
 This coenzyme attachment, --CH=N-, is called a Schiff base. a. Tyrosine to homogentisate (detection in
 Substrate-coenzyme Schiff base = aldimine and ketimine in urine is diagnostic for an enzyme
tautomeric equilibrium deficiency)
 Tautomer- ability of certain organic compounds to react in
isomeric structures that differ from each other in the position of
a hydrogen atom and a double bond
 Tautomerism Example
 Hydrolysis of the ketimine gives an alpha keto acid and
pyridoxamine. Figure 31. Homogentisation of Tyrosine
 Alpha-keto acid + amine group  new Schiff base.
4. Synthesis of Heme Precursors
 New amino acid… original schiff base.
a. Pyridoxal phosphate from succinyl CoA
converts Glycine to alpha-amino-beta
ketoadipic acid

Figure 32. Synthesis of Heme Precursor (alpha-amino-beta ketoadipic acid)

5. Component of Glycogen Phosphorylase

Figure 28. Tautomerization

 The ability of the schiff base to transfer electrons between
different atoms allows this coenzyme to participate in the
removal of other groups:
1. Decarboxylation of amino acids
2. Dehydrative deamination of serine and threonine
3. Desulfurative deamination of cysteine.
 The biochemical role of PP is associated with more than 60
enzymes involved in:
1. Synthesis of Neurotransmitters
a. Ex. Synthesis of dopamine through
carboxylation
Figure 33. Component of Glycogen Phosphorylase
6. Biosynthesis of Niacin from Tryptophan
a. Requires PP at portion of
hydroxykynurenine  hydroxyanthanilate
conversion
TRYPTOPHAN FORMYLKYNURENINE KYNURENINE
HYDROXYKYNURENINEPPHYDROXYANTHRANILATE
2-ACROLEYL-3-AMINOFUMARATENIACIN

7. Biosynthesis of Coenzyme A
Figure 29. Synthesis of Dopamine from Tyrosine
PANTOTHENATE + CYSTEINE PANTE-COENZYME A

PP—THEINE

BCHM Coenzymes 7 of 9
 8. Production of Ethanolamine and Taurine  Propionyl CoA carboxylase- for conversion of amino acids and
propionate to glucose in liver =Propionyl CoA + CO2 + ATP ----
methyl malonyl CoA + ADP + P

 Pyruvate carboxylase- for liver gluconeogenesis =Pyruvate +

CO2 + ATP ----oxaloacetate
 Biotin vs. Biocytin
9. Cofactor for Lysine Oxidase  Biocytin differs from biotin by the addition of a lysine group
a. Starts at a Lysine residue to produce the attached to the valeric acid side chain
crosslink
THF (Tetrahydrofolate)
 a folic acid-derivative coenzyme
 carries one-carbon groups on N5 and/or N10 and transfers
them to the substrate

 Folic Acid (vitamin B9)

 Pyridoxal Phosphate - Coenzyme required for the
 Occurs as polyglutamate derivatives with from 2 to 7
biosynthesis of another coenzyme
glutamic acid residues (n)
 to form Coenzyme A
 Taken up by intestinal mucosa cells and the extra glutamate
Biocytin residues removed by lysosomal conjugase
 derived from biotin (vitamin B7)  The free folic acid is reduced to tetrahydrofolate by
 structure which consists of an imidazole ring fused with dihydrofolate reductase and circulated in the plasma as N5-
thiophene ring with a valeric acid side chain methyl derivative.

Figure 35. Folate in the form of Polyglutamate derivative

 N5 – Methyltetrahydrofolate

Figure 34. Structure of Biotin, Biocytin, and Carboxy-biocytin

 Allows CO2 to become reactive: CO2 is relatively inactive with its

stable structure
 CO2 attaches to N of biocytin: Carbon becomes partially
positive and active
 The enzyme cannot utilize CO2 because the carbon is not Figure 36. Structure of cobalamine and N5 – Methyltetrahydrofolate
appreciably positive
 When biocytin is present, the carbon becomes positive and  one-Carbon groups
it can now be transferred in other reactions  Coenzyme in the synthesis of another molecule by one
 The enzyme methyl malonyl CoA carboxylase utilize this as a carbon group transfer
cofactor  Carbon groups transferred:
 It is a covalently bound prosthetic group of the enzymes
pyruvate carboxylase, acetyl CoA carboxylase and propionyl
CoA carboxylase
 Reactions involving Biotin
 Acetyl CoA carboxylase - for lipid synthesis from
acetate=Acetyl CoA + CO2 + ATP----methyl malonyl CoA + ADP
+P

Figure 37. carbon groups transferred

BCHM Coenzymes 8 of 9
 Figure 40. schematic presentation of cobalamin

CLINICAL APPLICATIONS:
 Megaloblastic Anemia of Vitamin B12 Deficiency
 the B12-dependent methionine synthesis reaction
(HOMOCYSTEINE + N5 –METHYL THF --- METHIONINE
+ THF) is the major pathway by which N5- METHYL THF can
return to the THF pool.
 in B12 deficiency, there is accumulation of N5-methyl THF
producing deficiency of THF derivatives.
 conversion of THF to its polyglutaminated form is also
inhibited
 Folate Trap & Pernicious Anemia
 vitamin B12 deficiency impairs folic acid metabolism leading
to functional folate deficiency which disturbs erythropoiesis
pernicious anemia (megaloblastic, immature precursor
RBCs).

Figure 38. One-carbon Group Transfer Mechanism

 De Novo Biosynthesis of Purine Rings

Figure 41. Homocysteine and the folate trap

Figure 39. De novo biosynthesis of purine rings mechanism

 Dihydrobiopterine Reductase (II) IV. REFERENCES

 Rodwell, Victor W; Bender, David A; Botham, Kathleen M; Kennelly, Peter J;
Weil, P. Anthony. (2018). Harper's Illistrated Biochemistry (31st ed.). United
States of America: McGraw-Hill Education.
 2022B, 2022C, 2022D trans
 Dr. Menorca’s lecture powerpoints

 Tetrahydrobiopterine (BPH4)
 coenzyme of phenylalanine hydroxylase.
 related to folic acid because of pteridine group.
 synthesized from GTP, not a vitamin
 dihydrobiopterine (bph2) produced during oxidation of
aromatic amino acid is reduced to BPH4 by a NAD-linked
dehydrogenase
 catecholamine synthesis is affected by BPH4 deficiency.

Cobalamin or Extrinsic Factor (Vitamin B12)

 Sub-Subheading
 consists of cobalt in a coordination state of six
 Schematic representation:

BCHM Coenzymes 9 of 9