Studies on the Binding of Thiamine Pyrophsophate 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 l0min at 25°C when 0.3μ_M TPP was incubated with 100μg of apodecarboxylase in a reaction mixture containing 20m_M Tris-maleate (pH 6.3) and 10m_M 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 oresence of Mn²⁺ and Mg²⁺ were 0.5μ_M and 25μ_M, respectively, and those for Mn²⁺ and Mg²⁺ in the presence of 1.2μ_M TPP were 0.29m_M and 2.0m_M, respectively. These results indicalte that the affinity of TPP binding to apodecarboxylase in markedly higher with Mn²⁺ than Mg²⁺. The role of metal ions in the binding is discussed.