Modeling an Elementary Step of the Enzyme Pyruvate Oxidase: Oxidation of a Thiamin Diphosphate-Bound Enamine Intermediate by a Flavin Analog

摘要

Subsequent to the first successful generation of enamines related to the central intermediate present on all thiamin diphosphate (ThDP)-dependent enzymes, we determined its structure in solution, its mechanism of oxidation at an electrode, the pK's leading to it both in DMSO and in water, and the kinetics of proton transfer to and from the C2α position. Herein is reported the mechanism of oxidation of the enaminne by a flavin adenine dinucleotide (FAD) analog, providing a model for the flavoenzyme pyruvate oxidase (POX), whose product is acetyl phosphate and CO_2. Scheme 1 shows the consecutive functions of the two coenzymes in this reaction. While several models already exist for this reaction, none to date has clarified the mechanism of oxidation of the enamine by flavin. The recent X-ray structure of POX suggests that the enzyme mediates transfer of the electrons from ThDP to FAD over a distance of 9.5 A.