On the alkylation of amino acid residues at the active site of ribonuclease
Abstract
Abstract Ribonuclease A has been subjected to reaction at pH 8.5 with a series of homologous α-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-α-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 119-position preferentially. No selectivity for the optical isomers of a given reagent is observed for the alkylation of lysine-41. Alkylation of the enzyme with iodoacetamide at pH 8.5 yields a complex mixture of products of which, in the initial stages of the reaction, the derivatives mono- and 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…
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