HIGHER-ORDER COMPLEX-FORMATION BETWEEN THE 72-KILODALTON TYPE-IV COLLAGENASE AND TISSUE INHIBITOR OF METALLOPROTEINASES-2

The collagenases are a class of matrix degradative enzymes whose actions are important in physiological and pathological processes. The human 72-kDa type IV collagenase (matrix metalloproteinase-2) and its proteinase inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2), are produced as a proenzyme-inhibitor complex by numerous cell lines. We analyzed the quaternary structure of and enzyme-inhibitor interactions in the native enzyme-inhibitor complex by studying the pattern of complexes demonstrated by molecular weight determination in nondenaturing polyacrylamide gels and evaluating the products formed by reaction of the native complexes with cross-linking agents. Electrophoresis in native polyacrylamide gels demonstrates that approximately 79% of the latent enzyme is present in a 1:1 bimolecular complex with the inhibitor TIMP-2, with 21% present as a complete tetrameric complex of two molecules of collagenase combined with two molecules of TIMP-2. The enzyme complex activated with organomercurials displays a shift to a higher proportion of the bimolecular complex with only 5% present as higher molecular weight complexes. Cross-linking of the latent and active forms of the complex with bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) tartarate demonstrates both the 1:1 and 2:2 complexes as well as an intermediate form that appears to be a complex composed of two molecules of collagenase and one of TIMP-2. The distribution of cross-linked products is unchanged with the addition of excess TIMP-2 to the reaction mix, implying that the binding sites for TIMP-2 to the initial enzyme-inhibitor complex are all occupied when the stoichiometry is 1 to 1. Cross-linking of the latent complex does not prevent organomercurial-induced activation and loss of an 80 amino acid prosegment. These cross-linked, activated complexes retain the ability to degrade gelatin in radiolabeled gelatin assays with the BS3-cross-linked, activated complex having a 7-fold higher specific activity than the un-cross-linked control. The BS3-cross-linked, activated complex is resistant to inhibition by free, exogenous TIMP-2. These results strongly suggest that there are two binding sites for TIMP-2 on collagenase and that binding to one site may decrease the affinity of binding of TIMP-2 to the second site.