Carboxymethylation of a ribonuclease from Rhizopus sp

In order to investigate the nature of the amino acid residues involved in the active site of a ribonuclease from Rhizopus sp. (RNase Rh), carboxymethylation of RNase Rh with iodoacetate was performed. RNase Rh was found to be inactivated markedly at pH 3-5 by iodoacetate. From the pH profile of the rate of inactivation of RNase Rh, it was suggested that functional groups having pKa values of ca. 7.3 and 4.3 might be involved in this inactivation reaction. The determination of the amino acid composition of RNase Rh inactivated by iodoacetate at pH 5.0 indicated that the formation of about one residue of N3-carboxymethylhistidine was responsible for the loss of enzymatic activity. The results were very similar to those for an RNase from Asp. saitoi having very similar base specificity. The carboxymethylation of RNase Rh was inhibited by competitive inhibitors. Thus, the histidine residue modified might be involved in the active site of the enzyme.