Kinetic analysis of the mechanism of interaction of full-length TIMP-2 and gelatinase A: Evidence for the existence of a low-affinity intermediate

We have undertaken a detailed analysis of the mechanism of inhibition of matrix metalloproteinase-2 (gelatinase A) by tissue inhibitor of metalloproteinase-2 (TIMP-2). Quenched fluorescent substrates have been used to analyze the rate of inhibition of gelatinase A by TIMP-2 over a wide range of TIMP-2 concentrations. When the values of the observed rate constant for the inhibition are plotted against TIMP-2 concentration, saturation is observed at high concentrations, providing evidence for formation of an intermediate in the pathway. Rate constants for the formation and dissociation of the intermediate are 5.9 x 10(6) M-1 s(-1) and 6.3 s(-1) respectively, giving a K-i for the initial step of approximately 1 mu M. The rate constant for the association of the final complex is 33 s(-1). By studying the dissociation of I-125-labeled TIMP-2 from a gelatinase A-TIMP-2 complex using ligand exchange experiments, we obtained a rate constant for the dissociation of the final stable complex of 2 x 10(-8) s(-1). This gives a value for the overall dissociation constant of approximately 0.6 fM.