ROLE OF LYS108 IN THE ENZYMATIC-ACTIVITY OF RNASE RH FROM RHIZOPUS-NIVEUS

In order to elucidate on the mechanism of action of RNase Rh from Rhiz opus niveus, we investigated the role of Lys108, which is conserved am ong the RNase T-2 family RNases except for two cases. The RNase activi ties of Lys108 mutant RNases, RNase RNAP K108R and K108L, are about 33 .5 and 3.1% of that of the wild type enzyme, respectively. The relativ e rates of cleavage of dinucleoside phosphates by these two mutant enz ymes were comparable to those with RNA as a substrate. The kinetic par ameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, Gr, U, and C) were measured. The data indicated that the K-m value s of the two mutant enzymes are similar to those of the wild-type enzy me. The rates of release of the four nucleotides from RNA by digestion with the mutant enzymes were in the order A>G>U>C, which is qualitati vely the same as that of the wild-type enzyme. From these data, we con cluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispens able for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition sta te as proposed in the case of Lys41 in RNase A, or may polarize the ph osphate moiety of the substrate.