Q Nguyen, F Willenbrock, MI Cockett, M O'Shea, AJ Docherty, G Murphy
The matrix metalloproteinases gelatinase A and stromelysin-1 have definable N-terminal (catalytic) and C-terminal domains. In order to analyze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the enzymes and the inhibitors were prepared in which the C-terminal domains had been deleted. Since the Ki values for TIMP inhibition of the matrix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activity assays. Rate constants for the association of the wild-type proteins were therefore determined and systematically compared with those for the deletion mutants. It was found that TIMP-1 binds more rapidly than TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzyme does not affect the rate of association of enzyme and inhibitor. This is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex formation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also comprised of an N- and C-terminal domain. By deletion mutagenesis, we found that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatinase A. Hybrids of the N- and C-terminal domains of gelatinase A and stromelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the interactions between the C-terminal domains of enzyme and inhibitor can occur irrespective of the nature of the N-terminal domain. We can conclude that the TIMPs have two major binding regions which associate in different ways with the domains of the enzymes gelatinase A and stromelysin-1. The N-terminal domains of the TIMPs bind to the enzyme catalytic domains to inhibit activity. The TIMP C-terminal domain acts to increase the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.