Alkylation and identification of the histidine residues at the active site of ribonuclease.

The reactions of iodoacetate and bromoacetate with ribonuclease have already provided some information on the relationship between the chemical structure and the catalytic a(*tivity of the enzyme. It is the purpose of this and the following paper to extend this information. In earlier studies, Gundlach, Stein, and Moore (1) showed that although alkylation at lysine, mrthioninc, or histidine residues could occur, the most specific inartivation by iodoacetate involved predominantly the formation at pH 5.5 to pH 6 of a derivative which contained a single residue of I (or 3)carboxymethylhistidinc. Independently, Rarnard and R. 11. Stein (2, 3) studied a similar type of reaction in which C%brled bromoacctate was the alkylating agent. From radioactivity measurements they concluded that one carboxymcthyl group per molecule of enzyme had been introduced. Upon oxidation of all of the protein in the reaction mixture, hydrolysis \yith chymotrypsin, and separation of the productas by paper electrophoresis, Rarnard and Stein found a single radioactive peptidc; from the amino acid composition of the peptide they concluded that the histidine which had been alkylated in ribonuclease was the one at position 119. Gundlach et al. also observed the formation at pH 5.5 of a small amount of a second inactive derivative which had a slower rate of travel on Amberlite IRC-50. The present communication is concerned with the cha.racterization of the two products of the reaction, the establishment of the position of the amino acid residues altered in each instance, and the determination of which of the two structural isomers (I-carboxymethylor 3-carboxymethylhistidine) is formed when a given imidazole ring in ribonuclease is alkylated by iodoacetate.