Document Detail


Role of Asp51 and Glu105 in the enzymatic activity of a ribonuclease from Rhizopus niveus.
MedLine Citation:
PMID:  8096846     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
K Ohgi; H Horiuchi; H Watanabe; M Iwama; M Takagi; M Irie
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of biochemistry     Volume:  113     ISSN:  0021-924X     ISO Abbreviation:  J. Biochem.     Publication Date:  1993 Feb 
Date Detail:
Created Date:  1993-05-13     Completed Date:  1993-05-13     Revised Date:  2007-12-19    
Medline Journal Info:
Nlm Unique ID:  0376600     Medline TA:  J Biochem     Country:  JAPAN    
Other Details:
Languages:  eng     Pagination:  219-24     Citation Subset:  IM    
Affiliation:
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Aspartic Acid / metabolism*
Base Sequence
Binding Sites / genetics
Catalysis
Glutamates / metabolism*
Glutamic Acid
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides
Rhizopus / enzymology*
Ribonucleases / genetics,  metabolism*
Sequence Homology, Amino Acid
Chemical
Reg. No./Substance:
0/Glutamates; 0/Oligodeoxyribonucleotides; 56-84-8/Aspartic Acid; 56-86-0/Glutamic Acid; EC 3.1.-/Ribonucleases

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