Oxalacetate Keto-Enol Tautomerase

Abstract

A protein that catalyzes the keto-enol tautomerism of oxalacetate was purified 56-fold by heat, acetone, and ammonium sulfate fractionation of a porcine kidney extract, followed by dialysis and column chromatography.This enzymatic activity was shown to be widely distributed.The tautomerase was separated from several other enzyme activities, including p-hydroxyphenylpyruvate keto-enol tautomerase.Several spectrophotometric assays for oxalacetate tautomerization were developed in the directions of both enolization and ketonization.The sensitivities of each system to changes in pH, ionic strength, and protein concentration were compared.The most useful method for following oxalacetate ketonization is the reaction coupled to the malate dehydrogenase-catalyzed reduction of the keto form of oxalacetate to malate.This coupled assay gives direct evidence for the keto form of oxalacetate as the substrate for malate dehydrogenase.By a similar coupled enzymatic reaction this conclusion has been extended to citrate synthase, glutamate-oxalacetate transaminase, and oxalacetate decarboxylase.A number of activators and inhibitors for the enzymatic tautomerization of oxalacetate are presented.This paper reports on a new and widely distributed catalytic activity of a protein that promotes the keto-enol tautomerism of oxalacetate.The search for such an enzymatic activity began when it was estimated that the rate of tautomerization in the neutral salt environment of the cell is at least an order of magnitude lower than the turnover of OAA' in its many enzymatitally catalyzed reactions.This estimate was based on the rate of enolization of OAA near neutral pH values reported earlier (1).The search for an OAA keto-enol tautomerase on this a priori basis was further encouraged by the realization that OAA isomers may be involved in the control of metabolic reactions of