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

• Published in: Biochemistry (Easton) Vol. 40; no. 34; pp. 10095 - 10102
• Main Authors: Reddick, Jason J, Nicewonger, Robb, Begley, Tadhg P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28.08.2001
• Subjects: Alkyl and Aryl Transferases - metabolism; Diphosphates - chemical synthesis; Diphosphates - metabolism; Indicators and Reagents; Kinetics; Oxygen Isotopes; Pyrimidines - chemical synthesis; Pyrimidines - metabolism; Substrate Specificity; Thiamine Monophosphate - metabolism; Thiazoles - chemical synthesis; Thiazoles - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010267q

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 [k cat(CH3) > 7800k cat(CF3)] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH3O-HMP-PP) is a good substrate [k cat(OCH3) > 2.8k cat(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.