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 Rhizopus niveus, we investigated the role of Lys108, which is conserved among the RNase T2 family RNases except for two cases. The RNase activities 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 relative rates of cleavage of dinucleoside phosphates by these two mutant enzymes were comparable to those with RNA as a substrate. The kinetic parameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, G, U, and C) were measured. The data indicated that the Km values of the two mutant enzymes are similar to those of the wild-type enzyme. 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 qualitatively the same as that of the wild-type enzyme. From these data, we concluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispensable for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition state as proposed in the case of Lys41 in RNase A, or may polarize the phosphate moiety of the substrate.
Ohgi
K
K
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
Iwama
M
M
Tada
K
K
Takizawa
R
R
Irie
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D005810:Multigene Family; D009154:Mutation; D009711:Nucleotides / Q000235:genetics; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship
D009711:Nucleotides; D004722:Endoribonucleases; C075842:ribonuclease Rh; D008239:Lysine