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

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degr adation. A Zn2+ metal, present in the active site, is involved in the catal ytic mechanism, and it is generally coordinated with histidyl and/or cystei nyl 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 degr ees C and 37 degrees C) on the enzymatic functional properties of the neutr ophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9) , using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximate to Leu-DPA-Ala-Arg-NH2. A global analysis of the observed proton-linked behav ior 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 w e 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 inves tigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays s ignificant differences with respect to what was previously observed for str omelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gr ay. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L . Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The fu nctional characterization of these enzymes can have some relevant physiolog ical significance, since it may be related to the marked changes in the env ironmental pH that collagenase and gelatinases may experience in vivo, movi ng from the intracellular environment to the extracellular matrix.