The Citric Acid Cycle

Summary:
Yields reduced coenzymes (NADH and QH2) and some ATP (2).
Preparative step is oxidative decarboxylation involving coenzyme A.
Occurs in eucaryotic mitochondrion and procaryotic cytosol.
How does the pyruvate get into the mitochondrion from the cytosol?
Pyruvate passes through channel proteins called porins (can transport
molecules < 10,000 daltons) located in outer mitochondrial membrane.
To get from intermembrane space to matrix involves pyruvate translocase
(symporter that also moves H+ into matrix).
CONVERSION OF PYRUVATE TO ACETYL COA
Enzyme is pyruvate dehydrogenase complex, composed of three enzymes:
1) pyruvate dehydrogenase
2) dihydrolipoamide acetyltransferase
3) dihydrolipoamide dehydrogenase
Reaction occurs in 5 steps:
1) E1 uses TPP as a prosthetic group and decarboxylates pyruvate --> forms
HETPP intermediate
2) E1 then transfers acetyl group to oxidized lipoamide --> acetyllipoamide
3) E2 transfers acetyl group to coenzyme A to form acetyl CoA;
dihydrolipoamide becomes reduced
4) E3 reoxidizes lipoamide portion of E2; prosthetic group of E3 (FAD)
oxidizes reduced lipoamide --> FADH2
5) NAD+ is reduced by E3-FADH --> E3-FAD + NADH + H+
E2 acts like a crane by swinging substrate between protein complexes in enzyme.
Regulation of PDH complex:
Regulated by covalent modification by phosphorylation.
inactive = phosphorylated; active = dephosphorylated
2
E1 inhibited at high [ATP]; inhibited at high [GTP]
activated by high [AMP], high [Ca2+], high [pyruvate]
E2 inhibited by high [acetyl CoA]
activated by high [CoA-SH]
E3 inhibited by high [NADH]
activated by high [NAD+]
THE CITRIC ACID CYCLE
Summary:
Composed of 8 reactions
4 carbon intermediates are regenerated
2 molecules of CO2 released (6C--> 4C)
Most of energy stored as NADH and QH2
1) citrate synthase
Irreversible reaction
Acetyl CoA reacts with oxaloacetate --> citrate and CoA
2) aconitase
Citrate --> isocitrate
3) isocitrate dehydrogenase
Irreversible reaction
Substrate first oxidized (2e- and H+ given to NAD+), then decarboxylated
Isocitrate --> -ketoglutarate + CO2 + NADH + H+
4) -ketoglutarate dehydrogenase complex
-ketoglutarate first decarboxylated, oxidized (2e- and H+ given to NAD+),
and HS-CoA added
Product is succinyl CoA
Enzyme complex similar the PDH, but has dihydrolipoamide
succinyltransferase instead of acetyltransferase.
5) succinyl CoA synthetase or succinate thiokinase
3
succinyl CoA --> succinate
Substrate has high energy thioester bond; that energy is stored as
nucleoside triphosphate via substrate level phosphorylation
GDP +Pi --> GTP mammals
ADP +Pi --> ATP plants and bacteria
6) succinate dehydrogenase complex
Enzyme is embedded in inner mitochondrial membrane.
Has FAD covalently bound to it (prosthetic group).
Converts succinate --> fumarate with generation of FADH2 --> ETS
FAD is regenerated by reduction of a mobile molecule called ubiquinone
(coenzyme Q) --> QH2.
7) fumarase
fumarate --> malate
8) malate dehydrogenase
L-malate --> oxaloacetate
2e- and H+ given to NAD+ --> NADH
Net reaction for citric acid cycle:
acetyl CoA + 3NAD+ + Q + GDP(ADP)+ Pi +2H2O ---> HS-CoA + 3NADH + QH2 +
GTP(ATP) + 2CO2 + 2H+
Energy Budget so far from 1 molecule of glucose:
glycolysis 2 ATP 2 NADH
Prep Step 2 NADH
TCA 2 ATP 6 NADH 2 QH2
4 ATP 10 NADH
ATP Production:
glycolysis 2 ATP 6 ATP equivalents
Prep Step 6 ATP equivalents
TCA 2 ATP 18 ATP equivalents + 4 ATP equivalents
4
4 ATP 34 ATP = 38 ATPs maximum
substrate (ox. phos.)
level phos.
REGULATION OF TCA CYCLE
There are 2 enzymes that are regulated:
1) isocitrate dehydrogenase
allosterically activated by high [Ca2+] and high [ADP]
allosterically inhibited by high [NADH]
2) -ketoglutarate dehydrogenase
allosterically activated by high [Ca2+]
allosterically inhibited by high [NADH] and high [succinyl CoA]
ENTRY AND EXIT OF METABOLITES
Citrate, -ketoglutarate, succinyl CoA, oxaloacetate lead to biosynthetic pathways.
Citrate --> fatty acids and sterols in liver and adipocytes
(cleaved into acetyl CoA if needed)
-ketoglutarate --> glutamate --> amino acid synthesis or nucleotide synthesis
succinyl CoA --> propionyl CoA --> fatty acid synthesis
--> porphyrin synthesis
oxaloacetate --> gluconeogenesis
--> asparate --> urea synthesis, a.a. synthesis, pyrimidine synthesis
Pathway intermediates must be replenished by anapleurotic reactions.