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Biophys J, October 2000, p. 2138-2149, Vol. 79, No. 4

pH- and Temperature-Dependence of Functional Modulation in Metalloproteinases. A Comparison between Neutrophil Collagenase and Gelatinases A and B

Giovanni Francesco Fasciglione,* Stefano Marini,* Silvana D'Alessio,dagger Vincenzo Politi,dagger and Massimo Coletta*

 *Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, I-00133 Roma, and  dagger PoliFarma, I-00155 Roma, Italy

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn2+ 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°C and 37°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-Glyapprox Leu-DPA-Ala-Arg-NH2. A global analysis of the observed proton-linked behavior for kcat/Km, kcat, and Km 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.

Biophys J, October 2000, p. 2138-2149, Vol. 79, No. 4
© 2000 by the Biophysical Society   0006-3495/00/10/2138/12  $2.00


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