The Identification of a Glutamic Acid Residue as Part of the Active Site of Ribonuclease T1

The experiments described in this communication were carried out in order to compare certain aspects of the chemistry of ribonuclease Tr from Aspergillus oryzae with those of bovine pancreatic ribormclease A. The primary structures of these enzymes of different specificities are known and are very dissimilar; ribonuclease A is a basic protein whereas ribonuclease Tr is acidic.Ribonuclease Tr, like the bovine enzyme, is inactivated by reaction with a HO-to 300-fold molar excess of iodoacetate at pH 5.5 and 37', but not by iodoacetamide.The time for 50% inactivation is 1 hour, which is considerably longer than that for ribonuclease A, and the rate of inactivation is maximal at pH 5.5.Use of W-labeled reagent has shown that inactivation is accompanied by the incorporation of 1 carboxymethyl group per molecule of enzyme.The reaction is inhibited by the presence of polyvalent anions such as citrate or phosphate, by metallic cations such as Cu++ or Znff, and by substrate analogues such as 2'-or 3'-guanylic acids.The inactivation reaction is also inhibited by 8 M urea and thus requires the native three-dimensional structure of the enzyme.Amino acid analyses of acid hydrolysates of the inactivated protein are indistinguishable from analyses of the parent enzyme; hence no reaction has occurred with residues of histidine or lysine which yield acid-stable carboxymethyl derivatives, and which are the sites of alkylation in pancreatic ribonuclease A. One equivalent of glycolic acid can be detected in the hydrolysate.The glycolic acid can be released from the protein by 1 M hydroxylamine at pH 9 in 8 M urea.Upon the working hypothesis that an ester had been formed on the carboxyl group of an aspartic or a glutamic acid residue, the aklylated protein was hydrolyzed by Nagarse to permit chromatographic isolation of a r4C-labeled peptide.The peptide proved to have been derived from Residues 57 to 62: Tyr-Glu-Trp-Pro-Ile-Leu.Hydrolysis of the peptide by aminopeptidase M yielded most of the expected amino acids (but no glutamic acid) plus a new ninhydrin-positive component which has been proved by synthesis to be the y-carboxymethyl ester of glutamic acid.These experiments