title: Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.
abstract: To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
authors.forename: K
authors.initials: K
authors.lastname: Katayanagi
authors.forename: K
authors.initials: K
authors.lastname: Morikawa
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
mesh_terms: D001483:Base Sequence; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D015183:Restriction Mapping; D016914:Ribonuclease H; D013816:Thermodynamics
chemical_list: D006146:Guanidines; D015345:Oligonucleotide Probes; D006639:Histidine; D004722:Endoribonucleases; D016914:Ribonuclease H; D019791:Guanidine