Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate.
Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine. Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM. A specific active-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation. 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster. Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (KI = 96 microM). Enzyme inactivation can be explained as the result of displacement of chloride from the chloropyrimidine by a nucleophile at the enzyme active site. The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage [Zoltewicz, J. A., & Kauffman, G. M. (1977) J. Am. Chem. Soc. 99, 3134-3142], which calls for the reversible nucleophilic addition of catalyst across the 1,6 double bond of thiamin's pyrimidine ring, may be applicable to thiaminase as well.
D019883:Alkyl and Aryl Transferases; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011743:Pyrimidines / Q000138:chemical synthesis / Q000494:pharmacology*; D014166:Transferases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D011743:Pyrimidines; C020859:2-amino-4-chloro-6-methylpyrimidine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022737:thiamin pyridinylase