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J. Biol. Chem., Vol. 281, Issue 50, 38302-38313, December 15, 2006

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The Roles of Substrate Thermal Stability and P₂ and P₁' Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity^*

Dmitriy Minond, Janelle L. Lauer-Fields, Mare Cudic, Christopher M. Overall, Duanqing Pei^¶, Keith Brew^||, Robert Visse^**, Hideaki Nagase^**, and Gregg B. Fields¹

From the Department of Chemistry and Biochemistry and ^||College of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431-0991, University of British Columbia Centre for Blood Research and the Canadian Institutes for Health Research Group in Matrix Dynamics, the Departments of Biochemistry and Molecular Biology, Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, the ^¶Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, and ^**Kennedy Institute of Rheumatology Division, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(279–523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(444–707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr²¹⁰ functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.

Received for publication, June 22, 2006 , and in revised form, October 20, 2006.

^* This work was supported by National Institutes of Health Grants AR 40994 (to K. B.), CA 98799, and EB 000289 (to G. B. F.), the Wellcome Trust Grant 057508 (to H. N.), and the Florida Atlantic University Center of Excellence in Biomedical and Marine Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431-0991. Tel.: 561-297-2093; Fax: 561-297-2759; E-mail: fieldsg{at}fau.edu.

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