ENZYME CATALYSIS AND REGULATION
Biochemical Characterization of the Catalytic Domain of Human Matrix Metalloproteinase 19: EVIDENCE FOR A ROLE AS A POTENT BASEMENT MEMBRANE DEGRADING ENZYME*

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We have recently cloned MMP-19, a novelmatrix metalloproteinase, which, due to unique structural features, was proposed to represent the first member of a new MMP subfamily (Pendás, A. M., Knäuper, V., Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and López-Otin, C. (1997) J. Biol. Chem. 272, 4281–4286). A recombinant COOH-terminal deletion mutant of MMP-19 (proΔ260–508MMP-19), comprising the propeptide and the catalytic domain, was expressed in Escherichia coli, refolded, and purified. Interestingly, we found that proΔ260–508MMP-19 has the tendency to autoactivate, whereby the Lys97-Tyr98 peptide bond is hydrolyzed, resulting in free catalytic domain. Mutation of two residues (Glu88 → Pro and Pro90 → Val) within the propeptide latency motif did not prevent autoactivation but the autolysis rate was somewhat reduced. Analysis of the substrate specificity revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH2 and, with higher efficiency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2. Kinetic analysis of the interactions of the catalytic domain of MMP-19 with the natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-based compounds efficiently inhibited the enzyme. The catalytic domain of MMP-19 was able to hydrolyze the basement membrane components type IV collagen, laminin, and nidogen, as well as the large tenascin-C isoform, fibronectin, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteinase capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrade triple-helical collagen. Finally, and in contrast to studies with other MMPs, MMP-19 catalytic domain was not able to activate any of the latent MMPs testedin vitro.

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This work was supported by Arthritis Research Campaign Grant K0541, by German Academic Exchange Service Studentship Grant D/97/2127, and by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.