Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Oct;79(4):2138–2149. doi: 10.1016/S0006-3495(00)76461-7

pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B.

G F Fasciglione 1, S Marini 1, S D'Alessio 1, V Politi 1, M Coletta 1
PMCID: PMC1301103  PMID: 11023917

Abstract

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the neutrophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.

Full Text

The Full Text of this article is available as a PDF (151.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aimes R. T., French D. L., Quigley J. P. Cloning of a 72 kDa matrix metalloproteinase (gelatinase) from chicken embryo fibroblasts using gene family PCR: expression of the gelatinase increases upon malignant transformation. Biochem J. 1994 Jun 15;300(Pt 3):729–736. doi: 10.1042/bj3000729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aimes R. T., Quigley J. P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem. 1995 Mar 17;270(11):5872–5876. doi: 10.1074/jbc.270.11.5872. [DOI] [PubMed] [Google Scholar]
  3. Antonini E., Ascenzi P. The mechanism of trypsin catalysis at low pH. Proposal for a structural model. J Biol Chem. 1981 Dec 10;256(23):12449–12455. [PubMed] [Google Scholar]
  4. Baramova E., Foidart J. M. Matrix metalloproteinase family. Cell Biol Int. 1995 Mar;19(3):239–242. [PubMed] [Google Scholar]
  5. Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., DeCarlo A., Engler J. A. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197–250. doi: 10.1177/10454411930040020401. [DOI] [PubMed] [Google Scholar]
  6. Blundell T. L. Metalloproteinase superfamilies and drug design. Nat Struct Biol. 1994 Feb;1(2):73–75. doi: 10.1038/nsb0294-73. [DOI] [PubMed] [Google Scholar]
  7. Bode W., Reinemer P., Huber R., Kleine T., Schnierer S., Tschesche H. The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity. EMBO J. 1994 Mar 15;13(6):1263–1269. doi: 10.1002/j.1460-2075.1994.tb06378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cha J., Auld D. S. Site-directed mutagenesis of the active site glutamate in human matrilysin: investigation of its role in catalysis. Biochemistry. 1997 Dec 16;36(50):16019–16024. doi: 10.1021/bi972223g. [DOI] [PubMed] [Google Scholar]
  9. Cossins J. A., Clements J. M., Ford J., Miller K. M., Pigott R., Vos W., Van der Valk P., De Groot C. J. Enhanced expression of MMP-7 and MMP-9 in demyelinating multiple sclerosis lesions. Acta Neuropathol. 1997 Dec;94(6):590–598. doi: 10.1007/s004010050754. [DOI] [PubMed] [Google Scholar]
  10. Dioszegi M., Cannon P., Van Wart H. E. Vertebrate collagenases. Methods Enzymol. 1995;248:413–431. doi: 10.1016/0076-6879(95)48027-7. [DOI] [PubMed] [Google Scholar]
  11. Fang J., Shing Y., Wiederschain D., Yan L., Butterfield C., Jackson G., Harper J., Tamvakopoulos G., Moses M. A. Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model. Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):3884–3889. doi: 10.1073/pnas.97.8.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fisher C., Gilbertson-Beadling S., Powers E. A., Petzold G., Poorman R., Mitchell M. A. Interstitial collagenase is required for angiogenesis in vitro. Dev Biol. 1994 Apr;162(2):499–510. doi: 10.1006/dbio.1994.1104. [DOI] [PubMed] [Google Scholar]
  13. Grams F., Reinemer P., Powers J. C., Kleine T., Pieper M., Tschesche H., Huber R., Bode W. X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design. Eur J Biochem. 1995 Mar 15;228(3):830–841. doi: 10.1111/j.1432-1033.1995.tb20329.x. [DOI] [PubMed] [Google Scholar]
  14. Harrison R. K., Chang B., Niedzwiecki L., Stein R. L. Mechanistic studies on the human matrix metalloproteinase stromelysin. Biochemistry. 1992 Nov 10;31(44):10757–10762. doi: 10.1021/bi00159a016. [DOI] [PubMed] [Google Scholar]
  15. Heppner K. J., Matrisian L. M., Jensen R. A., Rodgers W. H. Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol. 1996 Jul;149(1):273–282. [PMC free article] [PubMed] [Google Scholar]
  16. Hirose T., Reife R. A., Smith G. N., Jr, Stevens R. M., Mainardi C. L., Hasty K. A. Characterization of type V collagenase (gelatinase) in synovial fluid of patients with inflammatory arthritis. J Rheumatol. 1992 Apr;19(4):593–599. [PubMed] [Google Scholar]
  17. Holman C. M., Kan C. C., Gehring M. R., Van Wart H. E. Role of His-224 in the anomalous pH dependence of human stromelysin-1. Biochemistry. 1999 Jan 12;38(2):677–681. doi: 10.1021/bi9822170. [DOI] [PubMed] [Google Scholar]
  18. Johnson L. L., Pavlovsky A. G., Johnson A. R., Janowicz J. A., Man C. F., Ortwine D. F., Purchase C. F., 2nd, White A. D., Hupe D. J. A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition. J Biol Chem. 2000 Apr 14;275(15):11026–11033. doi: 10.1074/jbc.275.15.11026. [DOI] [PubMed] [Google Scholar]
  19. Knight C. G., Willenbrock F., Murphy G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett. 1992 Jan 27;296(3):263–266. doi: 10.1016/0014-5793(92)80300-6. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Maeda A., Sobel R. A. Matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol. 1996 Mar;55(3):300–309. doi: 10.1097/00005072-199603000-00005. [DOI] [PubMed] [Google Scholar]
  22. Massova I., Kotra L. P., Fridman R., Mobashery S. Matrix metalloproteinases: structures, evolution, and diversification. FASEB J. 1998 Sep;12(12):1075–1095. [PubMed] [Google Scholar]
  23. Mock W. L., Stanford D. J. Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism. Biochemistry. 1996 Jun 11;35(23):7369–7377. doi: 10.1021/bi952827p. [DOI] [PubMed] [Google Scholar]
  24. Morgunova E., Tuuttila A., Bergmann U., Isupov M., Lindqvist Y., Schneider G., Tryggvason K. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed. Science. 1999 Jun 4;284(5420):1667–1670. doi: 10.1126/science.284.5420.1667. [DOI] [PubMed] [Google Scholar]
  25. Nagase H., Fields G. B. Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers. 1996;40(4):399–416. doi: 10.1002/(SICI)1097-0282(1996)40:4%3C399::AID-BIP5%3E3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  26. Netzel-Arnett S., Sang Q. X., Moore W. G., Navre M., Birkedal-Hansen H., Van Wart H. E. Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin). Biochemistry. 1993 Jun 29;32(25):6427–6432. doi: 10.1021/bi00076a016. [DOI] [PubMed] [Google Scholar]
  27. Niyibizi C., Chan R., Wu J. J., Eyre D. A 92 kDa gelatinase (MMP-9) cleavage site in native type V collagen. Biochem Biophys Res Commun. 1994 Jul 15;202(1):328–333. doi: 10.1006/bbrc.1994.1931. [DOI] [PubMed] [Google Scholar]
  28. Rosenberg G. A., Dencoff J. E., Correa N., Jr, Reiners M., Ford C. C. Effect of steroids on CSF matrix metalloproteinases in multiple sclerosis: relation to blood-brain barrier injury. Neurology. 1996 Jun;46(6):1626–1632. doi: 10.1212/wnl.46.6.1626. [DOI] [PubMed] [Google Scholar]
  29. Stack M. S., Gray R. D. Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. J Biol Chem. 1989 Mar 15;264(8):4277–4281. [PubMed] [Google Scholar]
  30. Stack M. S., Gray R. D. The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase. Arch Biochem Biophys. 1990 Sep;281(2):257–263. doi: 10.1016/0003-9861(90)90441-z. [DOI] [PubMed] [Google Scholar]
  31. Stein R. L., Izquierdo-Martin M. Thioester hydrolysis by matrix metalloproteinases. Arch Biochem Biophys. 1994 Jan;308(1):274–277. doi: 10.1006/abbi.1994.1038. [DOI] [PubMed] [Google Scholar]
  32. Tschesche H. Human neutrophil collagenase. Methods Enzymol. 1995;248:431–449. doi: 10.1016/0076-6879(95)48028-5. [DOI] [PubMed] [Google Scholar]
  33. Vallee B. L., Auld D. S. Active-site zinc ligands and activated H2O of zinc enzymes. Proc Natl Acad Sci U S A. 1990 Jan;87(1):220–224. doi: 10.1073/pnas.87.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vu T. H., Shipley J. M., Bergers G., Berger J. E., Helms J. A., Hanahan D., Shapiro S. D., Senior R. M., Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 1998 May 1;93(3):411–422. doi: 10.1016/s0092-8674(00)81169-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walakovits L. A., Moore V. L., Bhardwaj N., Gallick G. S., Lark M. W. Detection of stromelysin and collagenase in synovial fluid from patients with rheumatoid arthritis and posttraumatic knee injury. Arthritis Rheum. 1992 Jan;35(1):35–42. doi: 10.1002/art.1780350106. [DOI] [PubMed] [Google Scholar]
  36. Weingarten H., Martin R., Feder J. Synthetic substrates of vertebrate collagenase. Biochemistry. 1985 Nov 5;24(23):6730–6734. doi: 10.1021/bi00344a064. [DOI] [PubMed] [Google Scholar]
  37. Welch A. R., Holman C. M., Huber M., Brenner M. C., Browner M. F., Van Wart H. E. Understanding the P1' specificity of the matrix metalloproteinases: effect of S1' pocket mutations in matrilysin and stromelysin-1. Biochemistry. 1996 Aug 6;35(31):10103–10109. doi: 10.1021/bi9601969. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES