α-Keto Acid Dehydrogenase Complexes

Pyruvate dehydrogenase, one of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, was rapidly inactivated by treatment with p-mercuribenzenesulfonate or fi-mercuribenzoate at pH '7.0.Similar results were obtained with free pyruvate dehydrogenase'and with pyruvate dehydrogenase bound to dihydrolipoyl transacetylase.Inactivation of pyruvate dehydrogenase was accompanied by modification of 2 sulfhydryl groups per molecule of enzyme (mol wt 183,000).Inactivation of the enzyme by the mercurial did not affect its state of aggregation or its ability to combine with the transacetylase.Thiamine pyrophosphate and Mg2f protected the enzyme against inactivation by the mercurial.Neither pyruvate nor Mg2+ was effective.In the presence of thiamine pyrophosphate and Mg2+, 2 -SH groups reacted with the mercurial without significant change in enzyme activity.Further treatment with the mercurial inactivated the enzyme and was accompanied by modification of 2 additional -SH groups.These results indicate that binding of thiamine pyrophosphate to the apoenzyme protected the two essential -SH groups and exposed, or enhanced the reactivity of, two nonessential -SH groups.The two essential -SH groups appear to be located either at the coenzyme binding sites or at other sites which, in turn, are modified by conversion of the apo-to the holoenzyme.