Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45.
The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006147:Guanine / Q000032:analysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009691:Nucleic Acid Denaturation; D006163:Ribonuclease T1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis*
D014443:Tyrosine; D006147:Guanine; D014364:Tryptophan; D004722:Endoribonucleases; D006163:Ribonuclease T1