Modification of the acetyl and nitro derivatives of carboxypeptidase a by N ‐bromoacetyl‐ N ‐methyl‐L‐phenylalanine

Glutamic acid 270 and tyrosine 248 have been implicated in the catalytic mechanism of carboxypeptidase A. The y-carboxylate of glutamic acid 270 is believed to function as nucleophile in the hydrolysis of peptide and ester substrates [l] . Modification of glutamic acid 270 either by Woodward reagent K [2,3] or by the affinity label, N-bromoacetyl-Nmethyl-Lphenylalanine (BrAcN(Me)Phe) [4,5] produces loss of enzymatic activity. The reaction of carboxypeptidase A with BrAcN(Me)Phe obeys saturation kinetics allowing separate estimation of the contributions of binding and reactivity (i.e. nucleophilicity and orientation) to the observed rate of inactivation. Modification of tyrosine 248 by several reagents, including N-acetylimidazole [7] produces enzyme derivatives with greatly reduced peptidase activity. Lipscomb has proposed that the phenolic hydroxyl of tyrosine 248 serves as proton donor in the hydrolysis of peptide substrates based both on the effect of chemical modification of tyrosine 248 and upon the location of this residue relative to the susceptible peptide bond as determined by X-ray crystallographic analysis [ 11. Alternatively, tyrosine 248 could function by orienting the substrate for nucleophilic attack by glutamic acid 270 rather than by direct participation in catalysis. The affinity labeling of the nitro and acetyl deriva-