2-Acetylthiamin pyrophosphate: an enzyme-bound intermediate in thiamin pyrophosphate-dependent reactions

Several enzymes catalyze reactions that may involve acetylthiamin pyrophosphate (acetyl-TPP) as an intermediate. These enzymes are phosphoketolase, pyruvate oxidase and several pyruvate oxidoreductases. Acetyl-TPP can be synthesized and used as a carrier to analyze quenched reaction mixtures for the presence of [14C]acetyl-TPP. Synthetic acetyl-TPP exhibits unusual chemical properties and a unique pH-rate profile that serves as a powerful means of characterizing [14C]acetyl-TPP that has been isolated from quenched enzymatic reaction mixtures. Using this and other methods, extensive evidence has been obtained for the involvement of acetyl-TPP in certain reactions catalyzed by the pyruvate dehydrogenase complex (PDH complex) of Escherichia coli. Acetyl-TPP is chemically competent as an intermediate in the decarboxylation and dehydrogenation of pyruvate by the PDH complex; and it is transiently formed during the course of this reaction. It may be an enzyme-bound intermediate or it may be in equilibrium with such an intermediate. Acetyl-TPP is very likely to be an intermediate of the phosphoketolase reaction. However, no direct evidence linking it to the phosphoketolase reaction mechanism is yet available. It is unclear whether acetyl-TPP is an intermediate in the pyruvate oxidoreductase reactions. In one example, that of the ketoacid oxidoreductase of Halobacterium halobium, analysis by electron paramagnetic resonance spectroscopy indicates the involvement of a hydroxyethyl-TPP-radical as an intermediate. It is unknown whether the subsequent reaction of this radical with coenzyme A an an oxidized FeS cluster to produce acetyl coenzyme A and the reduced cluster involves the intermediate formation of acetyl-TPP.