Open AccessIncrease in nucleolytic activity of ribonuclease T 1 by substitution of tryptophan 45 for tyrosine 45
Author(s) -
NISHIKAWA Satoshi,
MORIOKA Hiroshi,
KIMURA Tsuyoshi,
UEDA Yukari,
TANAKA Toshiki,
UESUGI Seiichi,
HAKOSHIMA Toshio,
TOMITA Kenichi,
OHTSUKA Eiko,
IKEHARA Morio
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14011.x
Subject(s) - rnase p , cyanogen bromide , biochemistry , mutant , guanine , chemistry , rnase h , microbiology and biotechnology , enzyme , biology , gene , nucleotide , peptide sequence , rna
The preparation and analysis of a mutant ribonuclease (RNase) T 1 which possesses higher nucleolytic activity than the wild‐type enzyme are described. The gene for the mutant RNase T 1 (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 T 1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild‐type RNase T 1 . 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 T 1 .