Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism.
Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-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 (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 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.
Reddick
J J
JJ
Department of Chemistry and Chemical Biology, 120 Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
Nicewonger
R
R
Begley
T P
TP
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D011756:Diphosphates / Q000138:chemical synthesis / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D010103:Oxygen Isotopes; D011743:Pyrimidines / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity; D013833:Thiamine Monophosphate / Q000378:metabolism*; D013844:Thiazoles / Q000138:chemical synthesis / Q000378:metabolism
C101901:4-amino-5-hydroxymethyl-2-methylpyrimidine; D011756:Diphosphates; D007202:Indicators and Reagents; D010103:Oxygen Isotopes; D011743:Pyrimidines; D013844:Thiazoles; D013833:Thiamine Monophosphate; D019883:Alkyl and Aryl Transferases; C435495:thiamin phosphate synthase