Kinetic properties of native and carboxy-peptidase-altered rabbit muscle aldolase.

In the accompanying publication (2), it is shown that the interaction of rabbit muscle aldolase and dihydroxyacetone phosphate yielded a product that could be visualized spectrophotometrically with properties consistent with an enzyme-substrate complex. Similar affinities were found for dihydroxyacetone phosphate with both native and carboxypeptidase-treated aldolase. It was of interest, therefore, to compare the apparent affinities of the two preparations for this substrate in the aldol condensation. A coupled reaction with cu-glycerophosphate dehydrogenase was devised to permit measurement of the kinetic constants of the condensation reaction of aldolase. The effect of carboxypeptidase treatment was previously noted by Drechsler, Boyer, and Kowalsky (3) as decreasing the rate of cleavage of fructose diphosphate by approximately one order of magnitude, although leaving the rate of cleavage of fructose l-phosphate and the Michaelis constants of these two substrates essentially unchanged. The results to be presented show that the altered enzyme reacts more efficiently with aldehydes than does the native enzyme. This finding and others, involving the effect of pH on the rate of reaction and the K, values of the hexose phosphates and the limitation of the rate of cleavage by aldehydes, define properties of aldolase that must be considered in analyzing its mechanism of action.