Enzymatic Thiamine Catalysis: Mechanistic Implications from the Three-Dimensional Structure of Transketolase

Based on the three-dimensional structure of transketolase, a mechanism for enzymatic thiamine catalysis is presented. At least three independent proton transfer steps occur during turnover and the catalytic groups involved have been identified. Deprotonation of the C2 carbon atom of the thiazolium ring, the first step of thiamine catalysis, is catalyzed by the 4′-imino group of the cofactor, formed after protonation of the N1′ nitrogen of the pyrimidine ring by Glu4l8. The 4′-NH2 group of the cofactor might also be involved in the second proton transfer step, occurring during the reaction of ThDP with donor substrate. More likely, however, this step is catalyzed by the side chain of His48l. The third proton transfer occurs during cleavage and formation of the addition compound of ThDP with substrate. The side chain of the conserved residue His3O is suggested to be involved in this proton transfer step.