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 MnSO
4. The relative ratio of the rates of TPP binding to apodecarboxylase in the presence of Mn
2+, Mg
2+, and Ca
2+ was 4, 1, and 0.2, respectively. The binding constants for TPP in the oresence of Mn
2+ and Mg
2+ were 0.5μ
M and 25μ
M, respectively, and those for Mn
2+ and Mg
2+ 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 Mn
2+ than Mg
2+. The role of metal ions in the binding is discussed.
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