Transition state analogs for thiamin pyrophosphate-dependent enzymes.

Abstract

Many of the transition states that are formed from thiamin pyrophosphate in enzymic reactions are expected to have structures in which the thiazolium ring of thiamin pyrophosphate has lost most of its positive charge. We have synthesized thiamin thiazolone pyrophosphate from the unphosphorylated compound. The sulphur-containing ring of thiamin thiazolone pyrophosphate is uncharged, and thus the compound resembles these transition states. In agreement with the prediction from the transition state theory of reaction rates, thiamin thiazolone pyrophosphate binds to Escherichia coli pyruvate dehydrogenase complex (EC 1.2.7.1) much more strongly than thiamin pyrophosphate itself. An upper limit for the value of the dissociation constant, calculated from the extent of inactivation of the enzyme by a low concentration of thiamin thiazolone pyrophosphate, is 5 X 10(-10) M at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. The dissociation constant for thiamin pyrophosphate under similar conditions is about 10(-5) M. The kinetics of inactivation of pyruvate dehydrogenase complex by thiamin thiazolone pyrophosphate are first order with respect to both enzyme and thiamin thiazolone pyrophosphate; the value of the second order rate constant is 5.7 X 10(5) M-1 min-1 at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. An analysis of the decrease in the rates of inactivation caused by thiamin pyrophosphate indicates that thiamin thiazolone pyrophosphate binds at the coenzyme sites. We have also synthesized thiamin thiothiazolone pyrophosphate and obtained very similar results with this compound.