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. 2004 Feb 1;377(Pt 3):775–779. doi: 10.1042/BJ20031067

Catalytic- and ecto-domains of membrane type 1-matrix metalloproteinase have similar inhibition profiles but distinct endopeptidase activities.

Douglas R Hurst 1, Martin A Schwartz 1, Mohammad A Ghaffari 1, Yonghao Jin 1, Harald Tschesche 1, Gregg B Fields 1, Qing-Xiang Amy Sang 1
PMCID: PMC1223890  PMID: 14533979

Abstract

Membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) is a major collagenolytic enzyme that plays a vital role in development and morphogenesis. To elucidate further the structure-function relationship between the human MT1-MMP active site and the influence of the haemopexin domain on catalysis, substrate specificity and inhibition kinetics of the cdMT1-MMP (catalytic domain of MT1-MMP) and the ecto domain DeltaTM-MT1-MMP (transmembrane-domain-deleted MT1-MMP) were compared. For substrate 1 [Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2), where Mca stands for (7-methoxycoumarin-4-yl)acetyl- and Dpa for N -3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl], the activation energy E (a) was determined to be 11.2 and 12.2 kcal/mol (1 cal=4.184 J) for cdMT1-MMP and DeltaTM-MT1-MMP respectively, which is consistent with k (cat)/ K (M) values of 7.37 and 1.46x10(4) M(-1).s(-1). The k (cat)/ K (M) values for a series of similar single-stranded peptide substrates were determined and found to correlate with a slope of 0.17 for the two enzyme forms. A triple-helical peptide substrate was predicted to have a k (cat)/ K (M) of 0.87x10(4) M(-1).s(-1) for DeltaTM-MT1-MMP based on the value for cdMT1-MMP of 5.12x10(4) M(-1).s(-1); however, the actual value was determined to be 2.5-fold higher, i.e. 2.18x10(4) M(-1).s(-1). These results suggest that cdMT1-MMP is catalytically more efficient towards small peptide substrates than DeltaTM-MT1-MMP and the haemopexin domain of MT1-MMP facilitates the hydrolysis of triple-helical substrates. Diastereomeric inhibitor pairs were utilized to probe further binding similarities at the active site. Ratios of K (i) values for the inhibitor pairs were found to correlate between the enzyme forms with a slope of 1.03, suggesting that the haemopexin domain does not significantly modify the enzyme active-site structure.

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Selected References

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  1. Itoh Y., Takamura A., Ito N., Maru Y., Sato H., Suenaga N., Aoki T., Seiki M. Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J. 2001 Sep 3;20(17):4782–4793. doi: 10.1093/emboj/20.17.4782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Knäuper V., López-Otin C., Smith B., Knight G., Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996 Jan 19;271(3):1544–1550. doi: 10.1074/jbc.271.3.1544. [DOI] [PubMed] [Google Scholar]
  3. Lauer-Fields J. L., Broder T., Sritharan T., Chung L., Nagase H., Fields G. B. Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates. Biochemistry. 2001 May 15;40(19):5795–5803. doi: 10.1021/bi0101190. [DOI] [PubMed] [Google Scholar]
  4. Lauer-Fields J. L., Tuzinski K. A., Shimokawa K. i., Nagase H., Fields G. B. Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases. J Biol Chem. 2000 May 5;275(18):13282–13290. doi: 10.1074/jbc.275.18.13282. [DOI] [PubMed] [Google Scholar]
  5. Lauer-Fields Janelle L., Juska Darius, Fields Gregg B. Matrix metalloproteinases and collagen catabolism. Biopolymers. 2002;66(1):19–32. doi: 10.1002/bip.10201. [DOI] [PubMed] [Google Scholar]
  6. Lehti Kaisa, Lohi Jouko, Juntunen Minna M., Pei Duanqing, Keski-Oja Jorma. Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of membrane-type 1 matrix metalloproteinase. J Biol Chem. 2002 Jan 4;277(10):8440–8448. doi: 10.1074/jbc.M109128200. [DOI] [PubMed] [Google Scholar]
  7. Li H., Bauzon D. E., Xu X., Tschesche H., Cao J., Sang Q. A. Immunological characterization of cell-surface and soluble forms of membrane type 1 matrix metalloproteinase in human breast cancer cells and in fibroblasts. Mol Carcinog. 1998 Jun;22(2):84–94. [PubMed] [Google Scholar]
  8. Lichte A., Kolkenbrock H., Tschesche H. The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2. FEBS Lett. 1996 Nov 18;397(2-3):277–282. doi: 10.1016/s0014-5793(96)01206-9. [DOI] [PubMed] [Google Scholar]
  9. McCawley L. J., Matrisian L. M. Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol. 2001 Oct;13(5):534–540. doi: 10.1016/s0955-0674(00)00248-9. [DOI] [PubMed] [Google Scholar]
  10. Mori Hidetoshi, Tomari Taizo, Koshikawa Naohiko, Kajita Masahiro, Itoh Yoshifumi, Sato Hiroshi, Tojo Hideaki, Yana Ikuo, Seiki Motoharu. CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain. EMBO J. 2002 Aug 1;21(15):3949–3959. doi: 10.1093/emboj/cdf411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Morrison J. F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta. 1969;185(2):269–286. doi: 10.1016/0005-2744(69)90420-3. [DOI] [PubMed] [Google Scholar]
  12. Murphy G., Knäuper V. Relating matrix metalloproteinase structure to function: why the "hemopexin" domain? Matrix Biol. 1997 Mar;15(8-9):511–518. doi: 10.1016/s0945-053x(97)90025-1. [DOI] [PubMed] [Google Scholar]
  13. Ohuchi E., Imai K., Fujii Y., Sato H., Seiki M., Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997 Jan 24;272(4):2446–2451. doi: 10.1074/jbc.272.4.2446. [DOI] [PubMed] [Google Scholar]
  14. Overall Christopher M. Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol. 2002 Sep;22(1):51–86. doi: 10.1385/MB:22:1:051. [DOI] [PubMed] [Google Scholar]
  15. Park H. I., Ni J., Gerkema F. E., Liu D., Belozerov V. E., Sang Q. X. Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor. J Biol Chem. 2000 Jul 7;275(27):20540–20544. doi: 10.1074/jbc.M002349200. [DOI] [PubMed] [Google Scholar]
  16. Park Hyun I., Turk Benjamin E., Gerkema Ferry E., Cantley Lewis C., Sang Qing-Xiang Amy. Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26. J Biol Chem. 2002 Jul 15;277(38):35168–35175. doi: 10.1074/jbc.M205071200. [DOI] [PubMed] [Google Scholar]
  17. Pei D., Weiss S. J. Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J Biol Chem. 1996 Apr 12;271(15):9135–9140. doi: 10.1074/jbc.271.15.9135. [DOI] [PubMed] [Google Scholar]
  18. Riddles P. W., Blakeley R. L., Zerner B. Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination. Anal Biochem. 1979 Apr 1;94(1):75–81. doi: 10.1016/0003-2697(79)90792-9. [DOI] [PubMed] [Google Scholar]
  19. Roderfeld M., Büttner F. H., Bartnik E., Tschesche H. Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris. Protein Expr Purif. 2000 Aug;19(3):369–374. doi: 10.1006/prep.2000.1259. [DOI] [PubMed] [Google Scholar]
  20. Sang Q. X., Jia M. C., Schwartz M. A., Jaye M. C., Kleinman H. K., Ghaffari M. A., Luo Y. L. New thiol and sulfodiimine metalloproteinase inhibitors and their effect on human microvascular endothelial cell growth. Biochem Biophys Res Commun. 2000 Aug 11;274(3):780–786. doi: 10.1006/bbrc.2000.3212. [DOI] [PubMed] [Google Scholar]
  21. Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994 Jul 7;370(6484):61–65. doi: 10.1038/370061a0. [DOI] [PubMed] [Google Scholar]
  22. Sternlicht M. D., Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001;17:463–516. doi: 10.1146/annurev.cellbio.17.1.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tam Eric M., Wu Yi I., Butler Georgina S., Stack M. Sharon, Overall Christopher M. Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain. The ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage. J Biol Chem. 2002 Jul 26;277(41):39005–39014. doi: 10.1074/jbc.M206874200. [DOI] [PubMed] [Google Scholar]
  24. Yana I., Weiss S. J. Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases. Mol Biol Cell. 2000 Jul;11(7):2387–2401. doi: 10.1091/mbc.11.7.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. d'Ortho M. P., Will H., Atkinson S., Butler G., Messent A., Gavrilovic J., Smith B., Timpl R., Zardi L., Murphy G. Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem. 1997 Dec 15;250(3):751–757. doi: 10.1111/j.1432-1033.1997.00751.x. [DOI] [PubMed] [Google Scholar]

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