Enzymatic properties of serine 93 mutants of RNase Rh from Rhizopus niveus. A trial to alter the base preference of RNase Rh.
In order to investigate the effects of mutation of Ser93, a component of base recognition site (B2 site) of a base non-specific RNase from Rhizopus niveus, we prepared 10 mutant enzymes at this position, S93A, S93V, S93F, S93T, S93G, S93D, S93N, S93E, S93Q and S93R, and their enzymatic activities towards RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 3.5-75% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by the mutant enzymes, we estimated the base preference of B1 and B2 base recognition sites. The results indicated that mutation of Ser93 to Phe, Thr, Glu. Gln and Arg caused the B2 site of the enzymes to more cytosine base preference and Asp and Asn substitution caused more uracil base preference. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkage at the 5'-side of cytidine or uridine. The results seem able to convert a base non-specific RNase to a base specific one.
Sanda
Akihiro
A
Department of Hygienic Technology, College of Environmental Health, Azabu University; Kanagawa 229-8501, Japan.
Iwama
Masanori
M
Ohgi
Kazuko
K
Inokuchi
Norio
N
Irie
Masachika
M
D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012233:Rhizopus / Q000201:enzymology*; D012694:Serine / Q000378:metabolism*
D012694:Serine; D004722:Endoribonucleases; C075842:ribonuclease Rh