The role of carboxylic acid groups in the action of glucoamylase I

Abstract

Received 31 October 1972 1. Introduction Carboxylic acid groups have been shown to be pre- sent in the active sites of many enzymes, including pep- sin [l], /3-D-glucosidase [2], lysozyme [3] and carboxy- peptidase A [4], and their presence has been suggested in many other cases [5]. Often the first indication that a carboxylic acid group is involved at the active site of an enzyme is obtained from the pH-rate profile, an ion- izable group with pK, about 3-5 being frequently as- sumed to be a carboxyl. Conclusions based on pH-stud- ies, however, must always be confirmed in other ways. A variety of methods have been used for the chemical modification of carboxyl groups in proteins and these can be valuable in structure-function correlations. For example, p-bromophenacyl bromide [6] and l-diazo- 6-phenyl-3-toluenesulphonamidobutan-2-one [7] inac- tivate pepsin by forming esters with the side-chain car- boxy1 of an essential aspartyl residue, and conduritol Bepoxide inactivates f?-D-glucosidase [8] by reaction with a carboxyl at its active site. Hoare and Koshland [9] developed a method for the modification of carboxyl groups in proteins, which involves the reaction of glycine methyl ester in the pres- ence of a water soluble carbodiimide. We have studied the effect of this reaction on the activity of glucoamyl- ase I, an enzyme whose pH-rate profile had suggested the possibility that a carboxyl group might be impli- cated in the enzymic process. 2. Materials and methods Crude glucoamylase from Aspergillus