ENZYMATIC-PROPERTIES OF MUTANT FORMS OF RNASE RH FROM RHIZOPUS-NIVEUSAS TO ASP51

In order to determine the role of Asp51 of RNase Rh from Rhizopus nive us, enzymes with mutations at the 51st position, D51N, D51E, D51Q, D51 S, D51T, D51A, and D51K, were prepared, and their enzymatic properties were investigated as to specific activity and base specificity. All t he mutant enzymes showed relatively high activity toward poly I and po ly C, and markedly reduced activity toward poly A and poly U. In parti cular, the enzymatic activities toward poly I of D51T and D51S were hi gher than that of RNase RNAP Rh. Among the mutant enzymes, D51N, D51S, and D51T showed more than ca. 30% of the activity of RNase Rh, when R NA, poly I and poly C were used as substrates, respectively. The subst itution of Ala, Glu, or Lys at Asp51 is unfavorable for enzymatic acti vity. Among XpGs (X=A, G, U, or C), D51N, D51S, and D51T showed higher activity toward GpG then CpG. Therefore, Asp51 in RNase Rh plays a cr itical role in the adenylic acid preference of RNase T-2 family enzyme s. Our results obtained with a protein engineering technique provide b asic insights into the control of the base specificity of RNase Rh.