07 Coenzymes

What is an enzyme?

A biological catalyst that accelerates chemical reactions without being consumed; binds substrate at an active site and forms an ES complex.

What is an apoenzyme?

The protein portion of a conjugated enzyme that is inactive without its cofactor/coenzyme.

What is a cofactor vs a coenzyme?

Cofactor = non-protein portion (inorganic or organic). Coenzyme = organic cofactor (often vitamin-derived) that helps transfer functional groups.

What is a holoenzyme?

The active enzyme: apoenzyme (protein) + cofactor/coenzyme (non-protein).

List the major enzyme classes.

Oxidoreductases; Transferases; Hydrolases; Lyases; Isomerases; Ligases; Translocases (added 2018).

What does the first digit of an EC class indicate?

It corresponds to the enzyme class (e.g., 1 = oxidoreductases).

General mechanism of coenzyme action?

Coenzyme accepts functional group X from substrate 1, transfers X to substrate 2, and is regenerated at the end.

Which vitamin is NAD/NADP derived from?

Niacin (Vitamin B3)

Which vitamin is FAD/FMN derived from

Riboflavin (Vitamin B2).

Which vitamin is Coenzyme A derived from?

Pantothenic acid (Vitamin B5).

Which vitamin is Thiamine Pyrophosphate (TPP) derived from?

Thiamine (Vitamin B1).

Which vitamin is Pyridoxal Phosphate (PLP) derived from?

Pyridoxine (Vitamin B6)

Which vitamin is Biocytin derived from?

Biotin (Vitamin B7).

Which vitamin is Tetrahydrofolate (THF) derived from?

Folic acid (Vitamin B9)

Which vitamin is Cobamide coenzyme derived from?

Vitamin B12 (Cobalamin).

Which coenzymes are not derived from vitamins?

Coenzyme Q (ubiquinone), Lipoic acid, Tetrahydrobiopterin.

Which coenzymes are not vitamin derivatives?

Coenzyme Q (ubiquinone), lipoic acid, and tetrahydrobiopterin.

What defines redox vs non-redox coenzymes?

Redox coenzymes transfer electrons/protons (e.g., NAD, FAD, CoQ). Non-redox coenzymes transfer other groups (e.g., acyl, amino, one-carbon).

Name the main redox coenzymes listed.

NAD/NADP, FAD/FMN, Ubiquinone (CoQ), Tetrahydrobiopterin.

Name the main non-redox coenzymes listed.

TPP, Lipoic acid, CoA (acyl transfer); PLP (amino group transfer); Biocytin (carboxylation/CO2 transfer); THF (one-carbon transfer); Cobamide (alkyl/methyl transfer).

What are the three structural parts of a nucleotide?

Nitrogenous base (purine/pyrimidine), sugar (ribose/deoxyribose), and phosphate.

Which bases are purines and which are pyrimidines?

Purines: Adenine, Guanine. Pyrimidines: Cytosine, Thymine (DNA), Uracil (RNA).

Difference between Thymine and Uracil?

Thymine = methylated form of uracil (the methyl group differentiates thymine).

What bond links sugar to base in nucleotides?

N-glycosidic bond between C1 of sugar and nitrogen of the base.

What are phosphodiester bonds?

Bonds linking nucleotides from 5’→3’ between sugar and phosphate groups.

Describe NAD+ structure in brief.

A dinucleotide: AMP portion + nicotinamide pseudonucleotide (derived from niacin). The 2’-OH of AMP can be phosphorylated to give NADP+.

What is the active site on NAD for redox?

C4 of the nicotinamide pyridine ring accepts a hydride ion (H⁻ = 2e⁻ + 1H⁺) converting NAD+ → NADH + H⁺.

Which enzymes commonly use NAD?

Lactate dehydrogenase (LDH), malate dehydrogenase, and many dehydrogenases in carbohydrate metabolism.

What is the spectral change used to monitor NAD/NADH?

NAD absorbs at ~260 nm; NADH has a peak at 340 nm — increase at 340 nm indicates NAD+ reduction.

Why is NADP important?

NADP (reduced NADPH) is a reducing agent for biosynthetic (anabolic) reactions (e.g., fatty acid synthesis via G6PD pathway).

Describe FAD and FMN briefly.

Both derived from riboflavin (B2). FAD = AMP + FMN (isoalloxazine ring + ribitol + phosphate). FMN = isoalloxazine + ribitol + phosphate (no AMP).

Which atoms in the flavin ring are active sites?

N1 and N5 of the isoalloxazine ring accept electrons/protons (can form semiquinone intermediates).

What is a semiquinone radical in flavins?

A stable one-electron reduced intermediate formed when FAD/FMN accept electrons/protons one at a time.

What is the absorbance feature of oxidized flavins?

Oxidized FAD/FMNs absorb maximally ~450 nm; absorbance abd color decreases on reduction to FADH2/FMNH2.

Name FAD-dependent enzymes given.

Succinate dehydrogenase (TCA cycle, Complex II) and acyl-CoA dehydrogenase (β-oxidation).

Name FMN-dependent enzymes given.

L-amino acid oxidase (Amino acid metabolism (oxidative deamination))

What is Coenzyme Q (ubiquinone)?

A lipid-soluble electron carrier in the inner mitochondrial membrane: benzoquinone ring + isoprenoid tail (variable n); shuttles electrons between Complexes I/II → III.

How does CoQ accept electrons?

Accepts electrons/protons one at a time to form a semiquinone intermediate, then ubiquinol (CoQH2) when fully reduced.

What is the absorbance of ubiquinone?

Ubiquinone absorbs maximally at ~270–290 nm; absorption disappears when fully reduced (ubiquinol).

What is tetrahydrobiopterin (BH4)?

A non-vitamin-derived coenzyme synthesized from GTP; required for hydroxylation of aromatic amino acids (Phe→Tyr, Tyr→DOPA, Trp→5-HT precursors).

How is BH4 regenerated?

Dihydrobiopterin is reduced back to BH4 using NADPH (via a reductase). Defects can impair hydroxylation (relevant to PKU).

Give clinical relevance of BH4 deficiency?

Impaired Phe→Tyr conversion (deficiency in the enzyme phenylalanine hydroxylase) causes buildup of phenylalanine (Phenylketonuria (PKU)-like features); may cause neurodevelopmental problems and pigment issues.

What is Thiamine Pyrophosphate (TPP)?

The active coenzyme form of vitamin B1 (thiamine): substituted pyrimidine–thiazole ring with terminal pyrophosphate; active C2 on thiazole forms a carbanion nucleophile.

Thiamine deficiency causes what?

Beri - beri - impaired carbohydrate metabolism and presenting as either cardiac failure with edema (wet beriberi) or peripheral neuropathy with muscle weakness (dry beriberi).

Why is the C2 hydrogen of TPP acidic?

Electron withdrawal by adjacent positively charged nitrogen and sulfur in the thiazole ring makes C2 H acidic → formation of carbanion that acts as nucleophile.

Name three reactions that require TPP.

Pyruvate decarboxylase (non-oxidative decarboxylation), Transketolase (pentose phosphate pathway), Pyruvate Dehydrogenase complex (oxidative decarboxylation).

What is required for TPP activity?

Mg²⁺ is required; TPP is inactivated by thiaminase (found in raw fish).

Summarize the pyruvate decarboxylase reaction?

Pyruvate (3C) → loss of CO₂ → acetaldehyde (2C); TPP forms a hydroxyethyl-TPP intermediate and is regenerated after acetaldehyde release.

What does transketolase do?

Transfers a 2-carbon keto fragment from xylulose-5-phosphate to ribose-5-phosphate producing glyceraldehyde-3-phosphate + sedoheptulose-7-phosphate (important in Hexose Monophosphate Shunt (carbohydrate metabolism).

List coenzymes required for oxidative decarboxylation of pyruvate (PDH complex).

TPP, lipoic acid, CoA, FAD, NAD — coordinated across E1, E2, E3 enzymes to produce acetyl Coa

What is lipoic acid and how is it attached?

A sulfur-containing cofactor (6,8-dithiooctanoic acid) covalently bound via ε-amino group of a lysine (ε-N-lipoyl-L-lysine) on E2.

Lipoic Acid is a coenzyme for

a-KG Dehydrogenase (TCA) and Pyruvate Dehydrogenase

What is the role of lipoic acid in PDH?

Accepts hydroxyethyl from TPP as an acyl group, transfers the acetyl group to CoA, and is reoxidized by FAD.

Describe Coenzyme A (CoA) structure in brief.

Contains an adenosine 3'-phosphate (AMP) linked via 5’ pyrophosphate to a pantothenic acid + β-alanine moiety and a terminal Thioethanolamine (active thiol).

Co-enzyme A is a cofactor for:

Fatty acyl CoASH Synthase and the Pyruvate Dehydrogenase Complex

What is the active site of CoA and its chemical role?

The terminal thiol (-SH) becomes nucleophilic when deprotonated, forming thioesters (e.g., acetyl-CoA, palmitoyl-CoA) for acyl transfer.

How are fatty acids activated for β-oxidation?

Fatty acid + ATP → acyl-adenylate + PPi; CoA attacks acyl-adenylate → acyl-CoA (thioester); enzyme = acyl-CoA synthetase (requires ATP → AMP + PPi).

Summarize the PDH complex enzyme/coenzyme mapping.

E1 (pyruvate dehydrogenase) uses TPP; E2 (dihydrolipoyl transacetylase) uses lipoic acid & CoA; E3 (dihydrolipoyl dehydrogenase) uses FAD → transfers electrons to NAD+.

Outline the PDH overall electron flow?

Hydroxyethyl from pyruvate → TPP → lipoic acid (acyl) → CoA (forms acetyl-CoA). Lipoic acid reduced → reoxidized by FAD → FADH2 → transfers hydride to NAD+ → NADH + H+.

What is biotin / biocytin and its general role?

Biotin (vitamin B7) is activated and attached to lysine (biocytin) on carboxylases to transfer CO₂ (bicarbonate) in carboxylation reactions (e.g., pyruvate carboxylase, acetyl-CoA carboxylase).

What is the avidin–biotin interaction relevance?

Avidin (egg white) binds biotin tightly and can prevent biotin absorption; the biotin–avidin interaction is exploited in lab biotinylation techniques.

How is malonyl-CoA formed (key step in FA synthesis)?

Acetyl-CoA + CO₂ (via carboxybiotin on acetyl-CoA carboxylase) → malonyl-CoA; biotin is released and regenerated; formation of malonyl-CoA is rate-limiting for FA synthesis.

What is pyridoxal phosphate (PLP)?

Active coenzyme form of vitamin B6 (pyridoxine). Exists as pyridoxal-P (aldehyde) and pyridoxamine-P (amine). PLP forms a Schiff base with lysine on enzymes.

PLP is inhibited by what drug?

Isoniazid (anti-TB drug)

How does PLP participate in amino acid reactions?

Forms Schiff base (aldimine) with amino acids → stabilizes carbanionic intermediates to catalyze transamination, decarboxylation, racemization, and eliminations.

Give the transamination (PLP) mechanism key steps (compact).

1) PLP bound as aldimine to enzyme lysine; 2) incoming AA displaces lysine → forms external aldimine; 3) double bond shift → ketimine; 4) hydrolysis → releases α-ketoacid and converts PLP→PMP; 5) PMP transfers amino group to acceptor ketoacid → regenerates PLP.

What is a ping-pong (double-displacement) reaction in context of PLP transamination?

One product is released before the second substrate binds — e.g., α-ketoglutarate → glutamate step releases a product (pyridoxamine phosphate) before pyruvate binds to form alanine.

Name PLP-dependent reactions with clinical relevance?

Decarboxylation of glutamate → GABA (neurotransmitter); decarboxylation of histidine → histamine; INH (isoniazid) can cause B6 deficiency → neuropathy (B6 is antidote).

What is racemization of amino acids (PLP)?

Conversion of L-amino acid ↔ D-amino acid via PLP-stabilized carbanion intermediate (important mechanistically; rare biologically).

What is tetrahydrofolate (THF)?

Active coenzyme form of folic acid (vitamin B9): pteridine + p-aminobenzoate (PABA) + glutamate(s); carries one-carbon units at different oxidation states (methyl, methylene, formyl, etc.).

Which one-carbon transfers does THF do?

Transfers -CH₃ (methyl), -CH₂- (methylene), -CHO (formyl), and related forms for nucleotide and amino-acid metabolism (e.g., serine↔glycine, thymidylate synthesis).

Describe serine ↔ glycine interconversion?

Serine donates a methylene group to THF (forms methylene-THF) to become glycine; reaction reversible and uses serine hydroxymethyltransferase.

Why is THF synthesis clinically important?

Folate antagonists (e.g., methotrexate, sulfonamides) inhibit THF formation → impair purine/pyrimidine synthesis → affect rapidly dividing cells (e.g., megaloblastic anemia).

What is cobamide (vitamin B12) coenzyme function?

Cobamide coenzymes (B12 derivatives) catalyze methyl transfer reactions (e.g., methylcobalamin in methionine synthase) and radical rearrangements (e.g., adenosylcobalamin).

Which forms of cobamide are biologically relevant?

Methylcobalamin and deoxyadenosylcobalamin (two coenzyme forms used in methyl transfers and radical chemistry, respectively).

Give 3 quick mnemonic anchors for coenzyme roles.

Redox carriers → NAD/FAD/CoQ; acyl carriers → CoA/lipoic acid/TPP (transfers aldehydes/acyls); one-carbon carriers → THF/B12/biocytin (carboxylation).

What spectral changes help measure enzyme activity for NAD and FAD?

NADH increase at 340 nm; oxidized FAD absorbance ~450 nm decreases when reduced (useful for FAD-dependent enzyme assays).

Which coenzyme deficiency causes beriberi?

Thiamine (B1) deficiency → impaired TPP-dependent enzymes (e.g., PDH, transketolase) leading to beriberi.

Which drug inactivates PLP and how is it treated?

Isoniazid (INH) can inactivate vitamin B6 → cause neuropathy; pyridoxine (vitamin B6) is the antidote.

Why is palmitate activation to palmitoyl-CoA energetically costly?

Requires ATP → AMP + PPi (equivalent of 2 high-energy bonds) to form acyl-adenylate then thioester with CoA.

Quick PDH clinical connection?

PDH dysfunction → build-up of pyruvate → lactic acidosis; PDH requires vitamins/coenzymes: B1 (TPP), B2 (FAD), B3 (NAD), B5 (CoA), lipoic acid.