title: Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant.
abstract: Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.
authors.affiliation: Biochemistry Department, Texas A & M University, College Station 77843.
authors.forename: B A
authors.initials: BA
authors.lastname: Shirley
authors.forename: P
authors.initials: P
authors.lastname: Stanssens
authors.forename: J
authors.initials: J
authors.lastname: Steyaert
authors.forename: C N
authors.initials: CN
authors.lastname: Pace
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics*; D005786:Gene Expression Regulation; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation
chemical_list: D005095:Exoribonucleases; C042081:exoribonuclease T