Mechanistic Studies on Thiamin Phosphate Synthase: Evidence for a Dissociative Mechanism

Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF3-HMP-PP) is a very poor substrate [kcat(CH3) > 7800kcat(CF3)] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH3O-HMP-PP) is a good substrate [kcat(OCH3) > 2.8kcat(CH3)] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (SN1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.