Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus.
In order to establish the role of Tyr57 and Trp49 in the enzymatic reaction of RNase Rh, several mutant enzymes at Tyr57 and Trp49 were prepared by protein engineering and their enzymatic properties were investigated. Among the four mutant enzymes at Trp49 (W49F, W49Y, W49A, and W49I), W49F showed 16% of the activity of the native enzyme, but the others (W49Y, W49A, and W49I) showed greatly decreased activity. The data showed that Trp49 is very important for the enzyme activity. Among 8 mutant enzymes at the 57th position, Y57F and Y57W showed similar enzymatic activity toward RNA to that of the wild-type enzyme, but the others (Y57G, Y57A, Y57V, Y57M, and Y57K) are more active toward RNA and less active toward XpGs. The reason for the apparent increase for RNA activity is discussed from the view point of substrate inhibition. It is noteworthy that W49F and Y57W became more pyrimidine base- and purine base-preferential, respectively.
Department of Microbiology, Hoshi College of Pharmacy, Shinagawa-ku, Tokyo.
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Iwama
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Irie
D000595:Amino Acid Sequence; D001483:Base Sequence; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D009711:Nucleotides; D012331:RNA, Fungal; D014443:Tyrosine; D014364:Tryptophan; D004722:Endoribonucleases; C075842:ribonuclease Rh