Role of Asp51 and Glu105 in the enzymatic activity of a ribonuclease from Rhizopus niveus.
The active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and Glu105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that Glu105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.
Ohgi
K
K
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
Horiuchi
H
H
Watanabe
H
H
Iwama
M
M
Takagi
M
M
Irie
M
M
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012233:Rhizopus / Q000201:enzymology*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D018698:Glutamic Acid; D012260:Ribonucleases