THE MECHANISM OF INHIBITION OF COLLAGENASE BY TIMP-1

Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight-bind ing inhibitor of fibroblast-type collagenase. Time-course data from in hibition experiments were analyzed by graphic analysis, by nonlinear r egression of the analytic integrals of the rate equations and by nonli near regression with numeric integration of the rate equations, With t he same assumptions, approximations and data, all three methods of ana lysis produced the same model preferences and values for the kinetic p arameters. The time course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncom petitive two-step mechanism, in which an inactive, rapidly formed, rev ersible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 com parable to that of collagenase, it is apparent that free TIMP-1 also f unctions in the breakdown of the tight complex, The rapidly formed com plex has a dissociation constant of 8 nM and reacts to the tight compl ex with a first-order rate constant of 0.003 s(-1). The back reaction of the tight complex to the rapid complex has a second-order rate cons tant of 5 x 10(4) M(-1) s(-1). The resulting global dissociation const ant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase conc entration. Collagenase without the carboxyl-terminal domain (mini-coll agenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 10(4) mol( -1) s(-1) and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s(-1). The apparent global dissociation cons tant for the tight complex (2.7 nM) is higher than that for the fibrob last-type collagenase, Participation of TIMP-1 in the dissociation is not demonstrable, Therefore, the carboxyl-terminal domain of fibroblas t type collagenase is important for the initial, rapid binding of TIMP -1 and the initial complex contributes to the overall binding.