Citric Acid Cycle (Krebs Cycle)

(KEGG pathway MAP00020)

The citric acid cycle, discovered in 1937 in animal tissue, is the central metabolic pathway for all aerobic processes. The cycle provides the complete oxidation of C2 units (acetyl-CoA) derived from fats, carbohydrates and lipids into carbon dioxide and water capturing the released energy as reductive power in the form of NADH and FADH2. The cycle also provides metabolic intermediates for biosynthetic purposes.

The enzymes and intermediates of the citric acid cycle are found inside the mitochondrial matrix, where the reductive power of NADH and FADH2 can directly be fed (in form of electrons) into the electron transport chain of the oxidative phosphorylation process in the inner membrane of this organelle. This electron flow is coupled to proton flow which is temporarily stored as an electrochemical gradient (proton motif force or PMF) which in turn is harvested by the ATP synthase to produce chemical energy.

The substrate of the cycle is an activated C2 unit - acetate in the form of Acetyl-Coenzyme A. It is derived from glycolysis by a decarboxylating dehydrogenase activity of pyruvate dehydrogenase. This mitochondrial enzyme is organized in three major protein complexes forming a particle larger than a ribosome.

Complex E1 E2 E3
Name Pyruvate dehydrogenase Dihydrolipoamide S-acetyltransferasee  Dihydrolipoamide dehydrogenase  
Subunits 24 subunits (EC & 24 subunits (EC 12 subunits (EC
Coenzyme Thiamin pyrophosphate (TPP) Lipoamide, Coenzyme A FAD, NAD
Reaction Oxidative decarboxylation of pyruvate to form actyl-TPP; release of carbon dioxide renders reaction irreversible Acetyl transfer via lipoamide to diffusible CoA FAD oxidizes lipoamide; FAD oxidized by NAD which is reduced to NADH/H+

The net reaction of pyruvate dehydrogenase is:

                pyruvate + CoA + NAD+  = acetyl-CoA + CO2 + NADH + H+

The energy contained is extracted by a cyclic set of reaction producing reducing equivalents and one molecule of GTP through a complete oxidation to two carbon dioxide molecules. The C2 unit in the form of an activated acetyl molecule, acetyl-CoA, is combined in a first step with the C4 unit oxaloacetate to form a C6 unit, citrate. Citrate is then oxidized and decarboxylated via a C5 intermediate (alpha-keto-glutarate) to oxaloacetate (C4). Starting from oxaloacetate, the full cycle adds two carbon units from acetate (CH3-COOH) and yields two carbon units as 2 CO2, 3 NADH, 1 GTP, and 1 FADH2 in form of chemical energy and 1 oxaloacetate. The cycle is non-reversible meaning that it cannot use CO2 for acetate formation. The following is a step-by-step description of the regeneration of oxaloacetate from citrate:

The cycle yields the following intermediates, some of which can be used as biosynthetic precursors.
Step   Formed by Energy yield
1. Citrate C6 Citrate synthase
2. Cis-aconitate C6 Aconitase
3. Iso-citrate C6 Aconitase
4. Alpha-ketoglutarate
C5 Isocitrate dehydrogenase (irreversible step) NADH
5. Succinyl-CoA (activated) C4 2-oxoglutarate dehydrogenase complex (irreversible step) NADH
6. Succinate C4 Succinyl-CoA synthetase GTP
7. Fumarate C4 Complex II (E.C.; membrane bound);succinate dehydrogenase FADH2
8. Malate C4 Fumarate hydratase
9. Oxaloacetate C4 Malate dehydrogenase NADH

The oxidation steps include lipoamide dehydrogenase (EC This enzyme is a component (subunit) of both multienzymes pyruvate dehydrogenase complex (E.Cs. + + and (E.Cs. + In fact, the usage of enzyme complex subunits in different enzymes with different substrate specificity but identical reaction mechanism (i.e., use of coenzymes), is commonly found in biological systems.

Biosynthetic function and control of citric acid cycle

As for glycolysis and gluconeogenesis, the citric acid cycle is regulated by the energy needs of the cell, mainly the rate of oxidative phosphorylation to replenish cellular ATP. Therefore, ATP and NADH exert a negative feed back control on citrate synthase (the first step in the cycle that feeds acetyl-CoA into it) and iso-citrate dehydrogenase, the first step in generating reductive power. This two allosteric regulations completely shut down the cycle when ATP synthesis is no longer needed and NADH starts to accumulate. Because both the Krebs cycle and oxidative phosphorylation are localized in the mitochondrial matrix, there is an immediate diffusion-controlled feed back mechanism to the key enzymes in both pathways. In addition, this allosteric control shuts down the cycle under anaerobic conditions, where NADH and FADH2 can no longer be oxidized by the electron transport chain components (complexes I & II).

A second regulatory mechanism is the feed back control of citrate on glycolysis and fatty acid synthesis. High citrate concentrations are indicative of high acetyl-CoA levels slowing down glycolysis while accelerating fatty acid synthesis reduces acetyl-CoA levels. Excess oxaloacetate not needed in the Krebs cycle (e.g. anaerobic conditions) will then be funneled into gluconeogenesis. The absolute concentration of oxaloacetate is the major focal point in metabolic coordination between energy household and biosynthetic demands by linking the metabolisms of carbohydrates, lipids, and amino acids together.

Citric acid cycle in E.coli and plants

The citric acid cycle is highly conserved in all organisms. There are metabolic differences, however, under different circumstances. In E.coli the citric acid cycle switches to a branched, non-cyclic mode under anaerobic conditions. The cycle is 'broken' at the alpha-ketoglutarate dehydrogenase step and takes a reverse path to succinyl-CoA starting from oxaloacetate via malate/aspartate, fumarate, and succinate. The branched citric acid 'cycle' mode provides biosynthetic precursors succinyl-CoA and alpha-ketoglutarate.  Oxaloacetate itself is used up in this reaction and needs to be replenished from the combination of two acetate units produced in the complete oxidation of pyruvate. This is performed by the glyoxylate cycle in bacteria (and plants; in the organelles called glyoxysomes). In this cycle, isocitrate is split by isocitrate lyase into succinate and glyoxylate. The latter can be linked with acetyl-CoA to form malate and CoA in a reaction catalyzed by malate synthase. Acetyl-CoA is formed from ATP and acetate by a phosphoryl group transfer to form acetyl-phosphate and ADP. The activated acetate-P can be moved to a CoA to form acetyl-CoA and inorganic phosphate.

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