Kinetic Analysis of the Binding of Human Matrix Metalloproteinase-2 and -9 to Tissue Inhibitor of Metalloproteinase (TIMP)-1 and TIMP-2

Matthew W Olson,David C Gervasi,Shahriar Mobashery,Rafael Fridman

doi:10.1074/jbc.272.47.29975

Matthew W Olson, David C Gervasi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.47.29975

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Abstract

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to 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 with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain 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 corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM 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 single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.

Highlights

The gelatinases matrix metalloproteinase (MMP)-2 and MMP-9 are two members of the MMP1 family, a group of zincdependent endopeptidases known to hydrolyze many components of the extracellular matrix [1]
Human recombinant latent and active MMP-2 and MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were purified to homogeneity
We have carried out a comprehensive study to determine the kinetic parameters for the binding of TIMP-1 and TIMP-2 to the latent and active forms of MMP-2 and MMP-9

Summary

EXPERIMENTAL PROCEDURES

Buffers—Buffer B (10 mM sodium acetate (pH 4.5)), buffer W (7.8 mM NaH2PO4, 8 mM Na2HPO4 (pH 7.2), 137 mM NaCl, 0.1 mM CaCl2, 3 mM KCl, 1.5 mM KH2PO4, and 0.02% Tween 20), buffer C (50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 0.02% Brij-35), buffer HA (25 mM Tris (pH 7.5), 25 mM NaCl, and 0.02% Brij-35), buffer R (50 mM Tris (pH 7.5), 5 mM CaCl2, 0.01% Brij-35), and phosphate-buffered saline (10 mM NaPO4 (pH 7.2), 150 mM NaCl) were used. Gelatin-Agarose Chromatography of TIMP-Gelatinase Complexes— Pro-MMP-9 and pro-MMP-2 (200 pmol) were combined with TIMP-1 and TIMP-2 (600 pmol), respectively, in buffer C (final volume of 0.1 ml) and incubated for 40 min at 25 °C. These mixtures were applied to a gelatin-agarose column (0.1 ml) and equilibrated with buffer C, and FIG. The koff value of TIMP-1 and TIMP-2 for the MMP-2 (62 kDa) and MMP-9 (82 kDa) species were determined except that the enzyme and inhibitor were incubated at concentrations of 300 and 360 nM, respectively, for 1 h at 25 °C. The inhibition constants (Ki) were calculated by Ki ϭ koff/kon

RESULTS

NB NB

DISCUSSION

Enzyme kon koff