Studies on the binding of thiamine pyrophosphate to apoenzyme of yeast pyruvate decarboxylase.

Abstract

The kinetics of the binding of thiamine pyrophosphate (TPP) to apoenzyme ot partially purified yeast pyruvate decarboxylase [EC 4.1.1.1] have been studied. A quantitative and spectrophotometric determination procedure for the TPP binding step, which is independent of the subsequent coupled alcohol dehydrogenase [EC 1.1.1.1] reaction, has been described. The binding of TPP to apodecarboxylase occurred rapidly and reached equilibrium after 10 min at 25°C when 0.3 μM TPP was incubated with 100 μg of apodecarboxylase in a reaction mixture containing 20 mM Tris-maleate (pH 6.3) and 10 mM MnSO4. The relative ratio of the rates of TPP binding to apodecarboxylase in the presence of Mn2+, Mg2+, and Ca2+ was 4, 1, and 0.2, respectively. The binding constants for TPP in the presence of Mn2* and Mg+ were 0.5 μM and 2.5 μM, respectively, and those for Mn* and Mg* in the presence of 1.2 μM TPP were 0.29 mM and 2.0 mM, respectively. These results indicate that the affinity of TPP binding to apodecarboxylase is markedly higher with Mn+ than Mg*. The role of metal ions in the binding is discussed.