title: Evidence for the importance of weakly bound water for matrix metalloproteinase activity.
abstract: The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated. In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates. Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme. The former reflects the hydrophobic nature of the enzyme-substrate interaction and the effect can be corrected for by using the solvent to water partition coefficient of the substrate in the mixed solvent systems. The catalyzed hydrolysis of substrate, corrected for the effect of hydrophobic partitioning, was demonstrated to be sixth order in water for gelatinase A and third order in water for stromelysin 1. Variation in water concentration did not produce saturation even at concentrations close to 55.5 M. The results indicate that weakly bound water molecules are essential to mediate the interaction between substrate and enzyme. The sensitivity of these enzymes to water concentration could be an important mechanism for regulating catalytic activity in vivo.
authors.affiliation: Department of Biochemistry, Queen Mary & Westfield College, University of London, U.K.
authors.forename: F
authors.initials: F
authors.lastname: Willenbrock
authors.forename: C G
authors.initials: CG
authors.lastname: Knight
authors.forename: G
authors.initials: G
authors.lastname: Murphy
authors.forename: I R
authors.initials: IR
authors.lastname: Phillips
authors.forename: K
authors.initials: K
authors.lastname: Brocklehurst
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry; D018093:Gelatinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012997:Solvents; D013379:Substrate Specificity; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
chemical_list: C095852:(7-methoxycoumarin-4-ylacetyl)-prolyl-leucyl-alanyl-norvalyl-(3-(2,4-dinitrophenyl)-2,3-diaminopropanoyl)-alanyl-argininamide; C095851:7-methoxycoumarin-4-yl-acetyl-prolyl-leucyl-glycyl-leucyl-(3-(2,4-dinitrophenyl)-2,3-diaminopropanoyl)-alanyl-argininamide; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2