title: Studies on RNase T1 mutants affecting enzyme catalysis.
abstract: Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40----Thr, Glu58----Asp, Glu58----Gln, His92----Ala and His92----Phe. His92----Ala and His92----Phe mutants are inactive. On the basis of their kcat/Km values, the mutants Glu58----Asp and Glu58----Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40----Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme.
authors.affiliation: Institut für Kristallographie, Freie Universität Berlin, Federal Republic of Germany.
authors.forename: H P
authors.initials: HP
authors.lastname: Grunert
authors.forename: A
authors.initials: A
authors.lastname: Zouni
authors.forename: M
authors.initials: M
authors.lastname: Beineke
authors.forename: R
authors.initials: R
authors.lastname: Quaas
authors.forename: Y
authors.initials: Y
authors.lastname: Georgalis
authors.forename: W
authors.initials: W
authors.lastname: Saenger
authors.forename: U
authors.initials: U
authors.lastname: Hahn
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D006163:Ribonuclease T1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D006163:Ribonuclease T1