METHYLMALONYL ISOMERASE, II. PURIFICATION AND PROPERTIES OF THE ENZYME FROM PROPIONIBACTERIA

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Abstract
Methylmalonyl isomerase from propionibacteria has been purified by chromatography on a cellulose column and by ammonium sulfate fraction. The activity obtained was about 200 times greater than that of previously reported preparations. The isolated enzyme is resolved from "B12 coenzyme" and is reactivated by the addition of coenzyme. The enzyme is free of lactic dehydrogenase methylmalonyl -oxaloacetic transcarboxylase, malic dehydogenase, CoA deacylase, and propionyl CoA transferase. A spectrophotometric assay for the isomerase has been developed in which this enzyme is coupled with methylmalonyl-oxaloacetic transcarboxylase and malic dehydrogenase. Propionyl CoA transferase has been assayed spectrophoto-metrically by coupling it with isomerase, transcarboxylase, and malic dehydrogenase. The equilibrium constant, (succinyl CoA)/(methyl-malonyl CoA), was found to be 10.5 for the isomerase reaction at pH 70, and 25[degree]. The F[degree]298 is calculated to be -1.4 x 103 calories.
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