EVIDENCE FOR THE IMPORTANCE OF WEAKLY-BOUND WATER FOR MATRIX METALLOPROTEINASE ACTIVITY

The effects of organic cosolvents on the kinetic characteristics of tw o matrix metalloproteinases, gelatinase A and stromelysin 1, were inve stigated. In each case, addition of the cosolvent resulted in a decrea se in the apparent k(cat)/K-m for the catalyzed hydrolysis of fluoroge nic peptide substrates. Two factors were identified as being responsib le for this decrease in catalytic activity: hydrophobic partitioning o f 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 b y using the solvent to water partition coefficient of the substrate in the mixed solvent systems. The catalyzed hydrolysis of substrate, cor rected 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 satur ation even at concentrations close to 55.5 M. The results indicate tha t weakly bound water molecules are essential to mediate the interactio n between substrate and enzyme. The sensitivity of these enzymes to wa ter concentration could be an important mechanism for regulating catal ytic activity in vivo.