Studies on the Binding of Thiamine Pyrophsophate to Apoenzyme of Yeast Pyruvate Decarboxylase

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.