KINETIC-ANALYSIS OF THE BINDING OF HUMAN MATRIX METALLOPROTEINASE-2 AND METALLOPROTEINASE-9 TO TISSUE INHIBITOR OF METALLOPROTEINASE (TIMP)-1 AND TIMP-2

The dissociation constants (K-d) of tissue inhibitor of metalloprotein ase (TIMP)-1 and TIMP-2, for the active and latent forms of matrix met alloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies, SPR analysis shows biph asic kinetics with high (nM) and low (mu M) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- an d 82-kDa species), respectively, In contrast, binding data of TIMP-8 t o an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics wi th K-d values of 315 and 60 nM, respectively, This suggests that the C TD contains the high affinity binding site, whereas the catalytic doma in contains the low affinity site. Also, binding of TIMP-2 to pro-MMP- 2 is stronger at both the high and low affinity sites than the corresp onding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the im portance of the N-terminal prodomain. In addition, the K-d value of TI MP-1 for the MMP-2 62-kDa species is 28.6 nn at the high affinity site , yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP -1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics, This suggests that a s ingle binding event results in enzyme inhibition, The kinetic paramete rs for the onset of inhibition are fast (k(on) -10(5) M-1 s(-1)) with slow off rates (k(off) similar to 10(-3) s(-1)). The inhibition consta nts (K-i) are in the 10(-7)-10(-9) hr range and correlate with the val ues determined by SPR.