Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.