title: Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system--an active site-directed reaction.
abstract: Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer, C.E. and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 +/- 7 and 4 +/- 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 +/- 0.14 min-1. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.
authors.affiliation: Institute of Biochemistry, Humboldt University of Berlin, G.D.R.
authors.forename: G
authors.initials: G
authors.lastname: Hausdorf
authors.forename: K
authors.initials: K
authors.lastname: Krüger
authors.forename: G
authors.initials: G
authors.lastname: Küttner
authors.forename: H G
authors.initials: HG
authors.lastname: Holzhütter
authors.forename: C
authors.initials: C
authors.lastname: Frömmel
authors.forename: W E
authors.initials: WE
authors.lastname: Höhne
mesh_terms: D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D001881:Borates; D006861:Hydrogen Peroxide; D007700:Kinetics; D008433:Mathematics; D008715:Methionine; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology
chemical_list: D001881:Borates; D008715:Methionine; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases; D013381:Subtilisins