The Citric Acid Cycle

Citric acid -a natural preservative in citrus fruits -Accumulation not related to citric acid cycle activities

(1) Acetyl-CoA production

(2) Acetyl-CoA oxidation - Citric acid cycle - or Tricarboxylic acid (TCA) cycle - or Kreb cycle

Cellular respiration - Aerobic conditions

- Complete oxidation of glucose, amino acids and fatty acids to CO2 and H2O

- Three major stages:

(3) Electron transfer and oxidative phosphorylation

Entry of pyruvate to mitochondria

Inner membrane

- A link between glycolysis and TCA cycle - Involves an enzyme complex with 3 components (E1, E2, and E3) - Overall an “oxidative decarboxylation” reaction - Five cofactors are involved (NAD+ as the final oxidizing agent)

Production of acetyl-CoA from pyruvate

(Co-enzyme A)

Pyruvate Dehydrogenase (PDH) Complex - Consisting of 3 different enzymes (E1, E2, E3) - Several reaction steps preformed before a product is released - Avoids diffusion of intermediates and allows efficient metabolite channeling

E1: Pyruvate dehydrogenase

- Contains TPP (or called TDP) in active site:

1. Decarboxylation of pyruvate

E1: Pyruvate dehydrogenase

Lipoyllysine: A lipoic acid linked to a lysine residue in E2

2. Transfer of acetyl group to a lipoyllysine in E2

E2: Dihydrolipoamide acetyltransferase

(Fully reduced form)

Arsenite poisoning:

Transfer of acetyl group to co-enzyme A:

E3: Dihydrolipoamide dehydrogenase

- Contains FAD as a prosthetic group

1. Regeneration of lipoamide (oxidized form)

2. Regeneration of FAD (oxidized form)

- A link between glycolysis and TCA cycle - Involves an enzyme complex with 3 components (E1, E2, and E3) - Overall an oxidative decarboxylation reaction - Five cofactors are involved (NAD+ as the final oxidizing agent)

Production of acetyl-CoA from pyruvate

(Co-enzyme A)

Regulation of the pyruvate dehydrogenase complex

(1)

Regulation of the pyruvate dehydrogenase complex

(2)

The citric acid cycle

Mitochondria

Reactions and enzymes of the citric acid cycle

(1) Citrate synthase

(OAA)

Enzyme-bound intermediate

- An acetyl group transferase - No ATP requirement

(2) Aconitase

- an isomerase (mutase)

(3) Isocitrate dehydrogenase

- Catalyzes an oxidative decarboxylation

(4) α-Ketoglutarate dehydrogenase

- An enzyme complex with 3 components (E1, E2, and E3) - Catalyzes an oxidative decarboxylation

- Resembles pyruvate dehydrogenase in both structure and function:

TPP, Lipoate, FAD

(5) Succinyl-CoA synthetase

-Hydrolysis of the thioester succinyl-CoA -Named for the reverse reaction (ligation of succinate and CoA-SH) -Substrate level phosphorylation

- Nucleoside diphosphate (NDP) kinase catalyzes

GTP + ADP GDP + ATP

Synthetases vs synthase - Both types of enzymes are involved in joining substrates together - Synthase reactions do not require NTP (nucleotide triphosphates) - Synthetase reactions use NTP (ATP or GTP) as an energy source

Proposed mechanism of succinyl-CoA synthetase:

Malonate - A structural analog of succinate not present in cells - Strong inhibitor of succinate dehydrogenase - Effectively blocks the TCA cycle activities

(6) Succinate dehydrogenase

- an membrane-bound enzyme

Q = ubiquinone QH2 = ubiquinol

(7) Fumarase

- Hydration of fumarate

(8) Malate dehydrogenase

Fates of carbon atoms in the citric acid cycle:

ATP production from reduced co-enzymes

- Summary of citric acid cycle:

- Electron transport chain: oxidation of reduced co-enzymes and ATP production

Net profit of aerobic degradation of glucose (32 ATP)

5 ATP (ETC)

2 ATP

5 ATP (ETC)

15 ATP (ETC)

QH2 3 ATP (ETC)

2 ATP

Regulation of citric acid cycle

Pyruvate Dehydrogenase

Complex

Isocitrate Dehydrogenase

α-Ketoglutarate Dehydrogenase

NAD+, CoA-SH

Citrate Synthase

ATP, NADH, citrate, succinyl-CoA

ADP +

- The cycle is precisely regulated to meet the cellular needs for ATP

Citric acid cycle is not always a “cycle”

The intermediates are used for both catabolism and anabolism

Cataplerotic reactions - Depletion of citric acid

intermediates

Anaplerotic reactions - Filling up of citric acid

intermediates