Biochemistry - Citric Acid Cycle

Citric Acid cycle

Also known as Krebs cycle after its discoverer Sir Hans Krebs, and as the tri-carboxylic acid (TCA) cycle based on the structure of citric acid

-The CAC oxidizes a 2C acetyl group completely to CO2, producing energy directly as GTP, and indirectly as NADH and FADH2

-Central biochemical pathway, and is amphibolic (meaning in participates in Anabolism and Catabolism)

Stage 1

-Production of acetyl CoA from pyruvate

-3C pyruvate changed into 2C acetyl by complex enzyme known as pyruvate decarboxylase by an oxidative decarboxylation

-Energy is derived from the PDHase reaction in the form of NADH. The reaction combines with the acetyl product with a carrier, Coenzyme A, to make acetyl coA for entry into the CAC

Pyruvate Dehydrogenase

PDH is made of three enzyme units:

-E1: Pyruvate dehydrogenase

-E2: a transacetylase

-E3: another dehydrogenase

The PDH complex is made of multiple copies of each enzyme plus associated cofactors to form an assembly line

PDH Reaction

Pyruvate + CoASH + NAD+ ---> Acetyl-CoA + CO2 + NADH

-Decarboxylation + Redox rxn

-Produces 1 NADH molecule and CO2

1. Citrate synthase

Condensation reaction

-Adds 2C Acetyl CoA to 4C oxaloacetate to form 6C citrate

-Citrate synthase enzyme

-Acetyl CoA added to the alpha-carbon of oxaloacetate

2. Aconitase

Isomerization Reaction

-Citrate isomerized to isocitrate

-Aconitase is enzyme

-Occurs between C2 and C3: OH and H of C2 and C3 respectively switch positions with one another

-Dehydration followed by rehydration

3. Isocitrate Dehydrogenase

FIRST oxidative decarboxylation

-Reduces number of C's by 1

-Isocitrate converted to alpha-ketoglutarate by removing carbonyl group

-Isocitrate dehydrogenase is enzyme

-decarboxylation + Redox, as seen in production of Acetyl CoA

4. Alpha-ketoglutarate dehydrogenase

SECOND oxidative decarboxylation

-Final reduction in number of C's

-Converts 5C alpha-ketoglutarate to 4C succinyl-CoA

-Again produces CO2 and NADH

-alpha-ketoglutarate dehydrogenase complex is enzyme

Oxidative decarboxylations

-Reactions reducing number of carbons by 1

Dehydrogenases

-Oxidative decarboxylation enzymes producing NADH

Alpha-ketoglutarate dehydrogenase

-Enzyme complex using CoA, similar to activity of pyruvate dehydrogenase

5. Succinyl-CoA Synthetase

Substrate level phosphorylation

-Only direct energy producing step, making GTP

-GTP later converted to ATP

-Succinyl CoA converted to succinate

-Succinyl-CoA synthetase enzyme

6. Succinate Dehydrogenase

Dehydrogenation reaction

-Succinate oxidized to fumarate

-Reduces FAD to FADH2

-FADH2 used for later energy production

-Succinate dehydrogenase enzyme

7. Fumarase

Rehydration reaction

-Double bond rehydrated in fumarate to form malate

-Fumarase is enzyme

8. Malate Dehydrogenase

-Dehydrogenation reaction

-Malate dehydrogenated to regenerate oxaloacetate to begin cycle again

-Produces 3rd NADH for energy

-Malate dehydrogenase is enzyme

Summary of TCA cycle

Acetyl CoA-->2CO2+GTP+FADH2+3NADH

-2 oxidative decarboxylations (not including pyruvate dehydrogenase reaction)

-1 Substrate level phosphorylation

-4 dehydrogenase reaction

Site of Citric Acid Cycle

CAC is located in mitochondria

-All substrates, cofactors, enzymes and products kept within specific cellular compartment

-Places NADH and FADH2 within matrix, readily accessible to membrane structure of mitochondrion

Sum of one turn of CAC

2 CO2, 3 NADH, 1 FADH2, 1 GTP

-1 more NADH from Pyruvate dehydrogenase

Summing up energy

-One GTP is same as 1 ATP

-Each NADH ultimately represents 3 ATP and each FADH2 represents 2 ATP

-3 NADH x 3=9

-1 FADH2 x 2=2

-1 GTP =12 ATP per acetyl unit

Energy from 1 glucose molecule

-Glycolysis started the process, making 2 NADH and 2 ATP, as well as 2 pyruvate

-2 pyruvate converted to 2 Acetyl CoA via PDHase, making 2 more NADH molecules

-Then, 2 times 12 ATP per acetyl unit, since 2 Acetyl CoA were produced

-SUM: Glucose-->6CO2 + 38 ATP, which saves about 40% of input glucose energy

Key regulatory steps

Regulation occurs at steps affecting carbon skeleton

-ATP/ADP ratio

-ATP/AMP ratio

-NADH/NAD+ ratio

Pyruvate dehydrogenase regulation

-Regulated by phosphorylation

-Useful regulation point because phosphate comes from ATP, and is indicator of energy state of cell

-Active while DEphosphorylated

Regulation via energy state

Pyruvate dehydrogenase kinase stimulated by ATP and inhibited by ADP acting on E1 with E1-PO4 inactive

-If low ATP and high ADP, pyruvate dehydrogenase remains active, supplying acetyl CoA to CAC

Anaplerotic Reactions

Reactions to regenerate TCA intermediates

-Primary enzyme is pyruvate carboxylase, making oxaloacetate by substrate feedforward in liver and kidney

-Tissue specific because need to replenish one intermediate to supply whole CAC

Regulation by substrate flux

Pyruvate carboxylase reaction is controlled by the levels of pyruvate

-When pyruvate is high, glycolysis is active but CAC is not

-Therefore, make oxaloacetate to engage the CAC and start consuming pyruvate in the form of Acetyl CoA

Citric acid cycle is amphibolic

-CAC does more than just make energy. It makes useful compounds that are starting points for synthesis reactions

-Thus, CAC is both catabolic and anabolic, which is amphibolic

Amphibolic interchanges: Oxaloacetate

-Oxaloacetate: directly converted into aspartate and asparagine, which can be converted to pyrimidines.

-May also be converted to phosphoenolpyruvate, which then forms serine, glycine, cysteine, phenylalanine, tyrosine, tryptophan, or glucose

Amphibolic interchanges: Succinyl CoA

-Can be converted to porphyrins or heme

Amphibolic interchanges: alpha-ketoglutarate

-Converted to glutamate, which can be converted to glutamine, proline, arginine, or purines

Amphibolic interchanges: Citrate

Converted to fatty acids, sterols

Interactions with other pathways

CAC generates useful compounds that intersect with other pathways to extend amphibolic nature of these reacions

-Important uses include:

-Amino acid metabolism, including nitrogen use

-Nucleotide metabolism

-Fatty acid metabolism

-Most other pathways

CAC and ketone bodies

-Fatty acid converted to acetyl-coA, which forms ketone bodies (hepatocyte, acetoacetate, d-Beta-hydroxybutyrate, acetone)