The citric acid cycle, also known as the Kreb's cycle and the tricarboxylic acid cycle, is composed of eight enzymes that serve to metabolize pyruvate, which itself is the end product of glycolysis, the metabolic breakdown of glucose. Citrate synthase, the first enzyme of the cycle, cannot utilize pyruvate until it is converted to acetyl-CoA by pyruvate dehydrogenase. The citric acid cycle results in the production of more than 30 high-energy ATP molecules and several proton-donating NADH molecules.
Pyruvate dehydrogenase is actually a complex made up of varying subunits of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase. These enzymes are bound noncovalently to the complex and work in unison to produce acetyl-CoA for citrate synthase. The complex, located in the mitochondria, also contains several cofactors which are essential to its function: thiamine pyrophosphate, lipoic acid, coenzyme A, flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD).
Pyruvate dehydrogenase complexes in E. coli are roughly 4600 kilodaltons in size and about 300 angstroms in diameter. Some eukaryotic complexes are 10000 kilodaltons in size and are composed of more than 50 enzymatic subunits. These are the largest known multienzyme complexes.
The processing of pyruvate into acetyl-CoA is accomplished through the assistance of the cofactors present in the complex. Thiamine pyrophosphate associates with pyruvate dehydrogenase and assists in the decarboxylation of pyruvate. Lipoic acid is linked to a lysine residue on dihydrolipoyl transacetylase; this prosthetic group accepts the acetyl group passed along by pyruvate dehydrogenase. Coenzyme A is the substrate for dihydrolipoyl transacetylase, and it accepts the acetyl group from lipoic acid. FAD and NAD are required to accept the protons which are the byproducts of the complex. A total of five reactions occur among the three enzymes and the five cofactors.
The pyruvate dehydrogenase complex is controlled in two ways. First, NADH (a product of the citric acid cycle) and acetyl-CoA (the product of pyruvate dehydrogenase) compete for the binding sites of coenzyme A and NAD in the complex. Thus, high levels of these two products inhibit pyruvate dehydrogenase so that acetyl-CoA is not being produced unnecessarily. The second method of control is by phosphorylation, which only occurs in eukaryotic cells. NADH and acetyl-CoA indirectly activate pyruvate dehydrogenase kinase, the phosphorylating agent which deactivates the first enzyme of the complex. Both insulin and calcium ions (a signal for increasing energy need) activate pyruvate dehydrogenase phosphatase, and the loss of phosphorylation reactivates the complex's first enzyme.