Enzymatic properties of glutamine 32 mutants of RNase Rh from Rhizopus niveus, a trial to alter the most preferential inter-nucleotidic linkages of RNase Rh.
In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5'-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.
Ohgi
Kazuko
K
Department of Applied Microbiology, Hoshi University, School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
Iwama
Masanori
M
Inokuchi
Norio
N
Irie
Masachika
M
D001483:Base Sequence; D001665:Binding Sites; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology*; D013379:Substrate Specificity; D015003:Yeasts
D015226:Dinucleoside Phosphates; D009711:Nucleotides; D012331:RNA, Fungal; D005973:Glutamine; D004722:Endoribonucleases; C075842:ribonuclease Rh