Enzymatic properties of double mutant enzymes at Asp51 and Trp49 and Asp51 and Tyr57 of RNase Rh from Rhizopus niveus.
Mutation of Asp51 of a base-nonspecific RNase, RNase Rh, to Ser, Thr, or Gln makes the enzyme more preferential for the dinucleoside phosphate (XpY) having G and C at the 5'-side (X). On the other hand the mutation of one of the B1 site components, Tyr57 to Trp, and Trp49 to Phe makes the enzyme more preferential for purine bases and pyrimidine bases, respectively. In this study, to obtain more specific RNases and RNases with different base specificity, we prepared double-mutant enzymes that have Ser, Thr, and Asn at the 51st position and Trp at the 57th position or Phe at the 49th position, and their enzymatic specificities were studied with XpYs as substrates. The double-mutant enzymes D51SY57W and D51TY57W are more guanylic acid preferential than the mother single-mutant enzymes, D51S and D51T, respectively. They are extremely guanylic preferential RNases. D51NY57W is more a guanylic acid preferential enzyme than D51N, but cytidylic acid preference is of a similar order to that of D51N. The double mutant enzymes D51NW49F and D51TW49F showed an increased cytidylic acid preference as well as guanylic acid preference as compared to the mother single-mutant enzymes, D51T and D51N. The results of analysis of base specificity by the release of mononucleotides from RNA and the rates of hydrolysis of homopolynucleotides led to the same conclusion as in the case of the hydrolysis of XpY.
Ohgi
K
K
Department of Microbiology, Hoshi College of Pharmacy, Tokyo, Japan.
Takeuchi
M
M
Iwama
M
M
Irie
M
M
D015226:Dinucleoside Phosphates / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D009711:Nucleotides; D012313:RNA; D004722:Endoribonucleases; C075842:ribonuclease Rh