Mechanism and Catalytic Site Atlas

Introduction

The TPP (thiamine diphosphate) cofactor is activated by the formation of an ionic N1'-H...O bond between the N1' atom of the aminopyrimidine ring of the cofactor and the OG of the intrinsic Glu59, resulting in the transfer of a negative charge from the protein to the cofactor. This transfer then imposes an active V-conformation that brings the N4' atom of the cofactor to the distance required for the intramolecular C2-H...N4' hydrogen bond with the thiazolium C2 atom. This initial activation is then followed by abstraction of the proton from the C2 atom. The resulting carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The puruvate is now covalently attached to the activated TPP. His271C donates a proton to the newly formed oxyanion. The covalently bound pyruvate undergoes decarboxylation and the carbanion formed initiates a nucleophilic attack on the second substrate (lipoamide) in an addition reaction with concomitant deprotonation of His128. Lipoamide is covalently bound to dihydrolipoamide acetyltransferase (the second catalytic component of the Pyruvate Dehydrogenase Complex) forming [dihydrolipoyllysine-residue acetyltransferase] lipoyllysine. His271C deprotonates the hydroxyl in an elimination which reforms the carbanionic form of ThDP and produces the N6-[(R)-S8-acetyldihydrolipoyl]-L-lysine residue product. Finally, the carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu59 and a bulk solvent molecule retruns His128 to its starting protonation state.