title: Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.
abstract: To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: C
authors.initials: C
authors.lastname: Katsuda
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: T
authors.initials: T
authors.lastname: Nakai
authors.forename: E
authors.initials: E
authors.lastname: Kitakuni
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: K
authors.initials: K
authors.lastname: Katayanagi
authors.forename: K
authors.initials: K
authors.lastname: Morikawa
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004229:Dithiothreitol; D004722:Endoribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D016914:Ribonuclease H; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D013438:Sulfhydryl Compounds; D004722:Endoribonucleases; D016914:Ribonuclease H; D004229:Dithiothreitol