THE EFFECT OF ENZYME ACETYLATION ON THE KINETICS OF THE CARBOXYPERTIDASE-A-CATALYZED HYDROLYSIS OF HIPPURL-L-BETA-PHENYLLACTIC ACID.

Abstract

One approach to the elucidation of the active site of an enzyme is to modify the site chemically, observe the effect of the chemical modification on the kinetics of the enzymatic process, and derive therefrom some conclusion about the binding and/or chemical reactions of the site. Many chemical modifications of enzymes have been effected, some of them quite specific and intimately connected with the active site. The most striking of these include chemical modifications which inhibit enzymatic activity completely, such as the classical inactivation of a-chymotrypsin and related enzymes by diisopropylphosphorofluoridatel and the inactivation of a-chymotrypsin by L-l-tosylamino-2-phenylethyl chloromethyl ketone.2 These modifications are usually interpreted in terms of the blocking of a specific chemical functionality of the active site. In addition to modifications resulting in complete inhibition, there exist a number of chemical modifications which result only in a partial change of elizymatic activity. These effects are more subtle, and their interpretation is not alway3 obvious without detailed investigation. Of many chemical modifications of chymotrypsin resulting in modified but finite reactivity, the following examples illustrate some of the complex behavioral patterns. The methionine residue, three amino acids from the reactive serine of the active site of chymotrypsin, on treatment with. hydrogen peroxide was converted to the corresponding sulfoxide. Thi chemical treatment resulted in a decrease of 10 per cent in Vmax and an increase of 350 per cent in Km(app).3 lodination of chymotrypsin (actually the above sulfoxide derivative) to the extent of 6.3 iodine atoms per molecule of chymotrypsin resulted in an essentially unchanged Vmax and a 6-fold increased Km (app).4 The methionine residue, three amino acids from the reactive serine of the active site of chymotrypsin, has also been alkylated by p-nitrophenyl bromoacetyl-a-aminoisobutyrate (presumably through the intervention of the corresponding acyl enzyme).5 This specifically derivatized enzyme shows a 10-fold increase in Km (app) and a 4-fold increase in Vmax toward a number of specific substrates of achymotrypsin.5, 6 One tentative conclusion from these data is that Km (app), which is related to binding, appears invariably to be adversely affected by modifications which do not entirely abolish activity. Another tentative conclusion from these data is that Vmax, which must reflect the catalytic process per se, may not be affected by such modifications, or may even increase! Vallee and co-workers have found that various treatments of carboxypeptidase A such as acylation with carboxylic acid derivatives, photooxidation, iodination, or replacement of the zinc ion of the enzyme by either cadmium or mercury ions increase the esterase activity of the enzyme, as measured by a standard assay using hippuryl-DL-3-phenyllactic acid or hippuryl-DL-f-indolyllactic acid, but decrease the peptidase activity, as measured by a standard assay using benzyloxycarbonyl-