On the alkylation of amino acid residues at the active site of ribonuclease.

Ribonuclease A has been subjected to reaction at pH 8.5 with a series of homologous cr-bromo acids and with iodoacetamide. At this pH, carboxyalkylation occurs predominantly at the 3 amino acid residues which have been implicated in the catalytic function of the enzyme: histidine-12, histidine-119, and lysine-41. The histidine reactions proceed at rates which vary from 3 to 25% of those observed at pH 5.5. The extent of reaction at the three positions is dependent upon the structure of the alkylating agent. In the reaction with bromoacetate, the derivative alkylated at lysine-41 accounts for the major proportion of the reaction products, while with D-a-bromo-n-butyrate, histidine-12 is the predominant site of alkylation. In general, the reactions at the histidine residues display the same stereospecificity toward a reagent at pH 8.5 as observed at pH 5.5 ; reagents of the D configuration favor reaction at histidine-12, while their L antipodes alkylate the 11%position preferentially. No selectivity for the optical isomers of a given reagent is observed for the alhylation of lysine-41. Alkylation of the enzyme with iodoaceramide at pH 8.5 yields a complex mixture of products of which, in the initial stages of the reaction, the derivatives monoand dicarboxamidomethylated at lysine-41 are prominent. The three products substituted at lysine-41 that have been characterized, namely the carboxymethyl, the carboxamidomethyl, and the dicarboxamidomethyl derivatives, all are essentially inactive. The active site histidine residues in the carboxymethyllysine-41 and carboxamidomethyllysine-41 derivatives react with bromoacetate at pH 5.5 at rates, respectively, 0.8 and 0.25 that of native ribonuclease. The effect of the II-substituent is in each case to depress the alkylation of histidine-12 in relation to that at histidine-11% These fmdings indicate that although the chemical modification of lysine-41 inactivates ribonuclease, it does not substantially inhibit the alkylation of the histidine residues which have been implicated as part of the active site of the enzyme. From the information currently available, a tentative working model has been proposed for the spatial relationships of the chemically reac-