FLUORESCENCE QUENCHING STUDIES OF MATRIX METALLOPROTEINASES (MMPS) - EVIDENCE FOR STRUCTURAL REARRANGEMENT OF THE PROMMP-2 TIMP-2 COMPLEX UPON MERCURIAL ACTIVATION/

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases whi ch are secreted from cells as zymogens and can be activated by treatme nt with organomercurial reagents or limited proteolysis. The proenzyme forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are found in c omplex with tissue inhibitor of metalloproteinases (designated proMMP- 2/TIMP-2 and proMMP-9/TIMP-1, respectively). The proposed mechanism of activation by mercurial compounds involves the induction of a conform ational change in the zymogen which leads to propeptide autoprocessing . To investigate the possibility of conformational differences in MMPs , solute quenching of MMP intrinsic fluorescence was used to probe the relative exposure of tryptophan residues in latent and mercurial-acti vated MMPs, Our data demonstrate that fluorescence quenching of the pr oMMP-2/TIMP-2 complex by either acrylamide or iodide is significantly increased following mercurial activation In contrast, no significant c hange in tryptophan accessibility accompanies mercurial treatment of e ither proMMP-2 or TIMP-8 alone, or mercurial-activated MMP-2 mixed wit h TIMP-2. To determine whether the enhanced fluorescence quenching was unique to the activated proMMP-2/TIMP-2 complex, similar experiments were performed using MMP-1, MMP-3, and MMP-9/TIMP-1 complex, In all ca ses, both latent and mercurial-treated MMPs exhibited similar fluoresc ence quenching profiles, suggesting that there are no significant conf ormational differences between the zymogen and activated forms of MMP- 1, -2, -3, or -9/TIMP-1. The enhanced fluorescence quenching observed with mercurial-treated proMMP-2/TIMP-2 is indicative of increased expo sure of a previously buried tryptophan residue(s), providing evidence for a structural rearrangement of the activated complex. These data, t ogether with our previous biochemical observation that mercurial treat ment of proMMP-2/TIMP-2 exposes the MMP-2 active site without propepti de processing (Y. Itoh et al. (1995) Biochem. J. 308, 645-651), sugges t that the activated proMMP-2 in the complex may represent a transitio nal conformational intermediate in MMP activation. (C) 1996 Academic P ress.