Evidence That Three Histidine Residues of a Base Non-Specific and Adenylic Acid Preferential Ribonuclease from Rhizopus niveus Are Involved in the Catalytic Function

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or HislOO showed less than 0.03%, but a mutant enzyme at HislO4 showed 0.54 % of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or HislO0 towards 2-AMP decreased twofold, but that at HislO4 decreased more markedly. There-fore, we assumed that these three histidine residues are components of the active site of RNase Rh, that HislO4 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, HislO4, and HislOO by comparison of the 'H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual p£