Studies on the Enzyme-Substrate Complex. II

The Reactions of Carboxypeptidase with Chloroacetyltyrosine by the Crossing Electrophoresis

Abstract

The formation of the enzyme-substrate complex of carboxypeptidase with its substrate, chloroacetyltyrosine was demonstrated by the crossing paper electrophoresis. The method consists in making the line of chloroacetyltyrosine drawn perpendicularly to the direction of the electrical field, encounter with that of carboxypeptidase drawn perpendicularly to the former. The line of substrate formed a groove at the point of crossing with the line of enzyme.
When the carboxypeptidase was inactivated by the addition of o-phenanthroline, the binding with the substrate occurred as in the case of active enzyme. When irreversibly inactivated in 40% alcohol, the carboxypeptidase did not bind with the substrate.
In the presence of the competitive inhibitor, ß-phenylpropionic acid, the binding of the carboxypeptidase with the substrate did not occur. But cinnamic acid and lactic acid did not inhibit the formation of the complex.