title: Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.
abstract: The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: C
authors.initials: C
authors.lastname: Katsuda
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D016914:Ribonuclease H; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D007461:Iodoacetates; D004228:Dithionitrobenzoic Acid; D004722:Endoribonucleases; D016914:Ribonuclease H; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid