Summary of the citric acid cycle

• For each acetyl CoA which enters the cycle:

(1) Two molecules of CO2 are released

(2) Coenzymes NAD+ and FAD (Q) are reduced

(3) One GDP (or ADP) is phosphorylated

(4) The initial acceptor molecule (oxaloacetate) is reformed

There are 8 reactions in the cycle. 1. Condensation – only rxn. that generates a C-C bond Enzyme: citrate synthase

•Binds substrates one at a time

•Induced fit (conformational change)

•Breaking high energy thioester bond makes -G

Acid/Base catalysis

• Claisen condensation

• Stabilization of intermediate

• Release of product

2. Rearrangement –Elimination, then addition of H2O

• Intermediate: C=C bond of cis-aconitate• Stereospecific• Reversible• Driven to completion by depletion of

product by next rxn. in the cycle

Enzyme: aconitase (aconitate hydratase)

3. Oxidative decarboxylation• Metabolically irreversible • One of four oxidation-reduction reactions of

the cycle• Hydride ion from the C-2 of isocitrate is

transferred to NAD+ to form NADH• Oxalosuccinate is decarboxylated• Mn2+ stabilizes intermediate

Enzyme: Isocitrate Dehydrogenase

2 forms – one uses NAD+; other uses NADP+

4. Oxidative decarboxylation

Enzyme: -Ketoglutarate Dehydrogenase Complex

Similar to pyruvate dehydrogenase complex (E3 is identical)

Same coenzymes, identical mechanisms

E1 - a-ketoglutarate dehydrogenase (with TPP)

E2 - succinyltransferase (with flexible lipoamide prosthetic group)

E3 - dihydrolipoamide dehydrogenase (with FAD)

5. Substrate level phosphorylation

• Free energy in thioester bond of succinyl CoA is conserved as GTP (or ATP)

Enzyme: Succinyl-CoA Synthetase

• Dimer

– One subunit specific for GTP or ATP

– GTP and ATP are energetically equivalent

GTP + ADP GDP + ATP

Enzyme: nucleoside diphosphate kinase

G0 = 0 kJ/mol

6. Dehydrogenation (oxidation)Enzyme: Succinate Dehydrogenase (SDH) Complex• Located on the inner mitochondrial

membrane (plasma membrane in prokaryotes)

• Dehydrogenation is stereospecific; only the trans isomer is formed

• Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters

• Electrons are transferred from succinate to ubiquinone (Q), a lipid-soluble mobile carrier of reducing power

• FADH2 generated is reoxidized by Q

• QH2 is released as a mobile product (ETC)

• Substrate analog malonate is a competitive inhibitor of the SDH complex

7. Hydration

• Stereospecific trans addition of water to the double bond of fumarate to form L-malate

Enzyme: fumarase (fumarate hydratase)

8. Oxidation

Enzyme: L-malate dehydrogenase

• Endergonic, but product constantly removed by highly exergonic rxn. #1 (citrate synthase)

• [oxaloacetate] < 10-6 M (very low)

• Fates of carbon atoms in the cycle

• Carbon atoms from acetyl CoA (red) are not lost in the first turn of the cycle

Energy conservation by the cycle

• Energy is conserved in the reduced coenzymes NADH, FADH2 (QH2) and one GTP (ATP)

• Reduced coenzymes can be oxidized to produce ATP by oxidative phosphorylation (electron transport chain, ETC)

Reduced Coenzymes Fuel the Production of ATP

• Each acetyl CoA entering the cycle nets:

(1) 3 NADH

(2) 1 FADH2 (QH2)

(3) 1 GTP (or 1 ATP)

In ETC further oxidation yields:

2.5 ATP per NADH

1.5 ATP per FADH2 (QH2)

Glucose degradation via glycolysis, citric acid cycle, and oxidative phosphorylation

From 1 glucose 30-32 ATP