THE ACTIVITY OF THE TISSUE INHIBITORS OF METALLOPROTEINASES IS REGULATED BY C-TERMINAL DOMAIN INTERACTIONS - A KINETIC-ANALYSIS OF THE INHIBITION OF GELATINASE-A

The cloning and expression of the full-length tissue inhibitor of meta lloproteinase 2 (TIMP-2), DELTA187-194TIMP-2, and DELTA128-194TIMP-2 a nd the purification of these inhibitors and a cleaved version of TIMP- 2 lacking nine C-terminal amino acids (DELTA186-194TIMP-2) are describ ed. The mechanism of inhibition of gelatinase A by the TIMPs was inves tigated by comparing the kinetics of association of TIMP-1, TIMP-2, th e C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatin ase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TI MP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal pep tide of TIMP-2 is proposed to exist as an exposed ''tail'' responsible for binding to progelatinase A and for increasing the rate of inhibit ion of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP- 1 and TIMP-2 participate in low-affinity interactions with the C-termi nal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.