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 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 approximate to Leu-DPA-Ala-Arg-NH2. 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.

Fasciglione G.F., M.S. (2000). pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B. BIOPHYSICAL JOURNAL, 79(4), 2138-2149.

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

FASCIGLIONE, GIOVANNI;MARINI, STEFANO;COLETTA, MASSIMILIANO
2000

Abstract

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 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 approximate to Leu-DPA-Ala-Arg-NH2. 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.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10
English
Con Impact Factor ISI
collagenase; gelatinase A; gelatinase B; matrix metalloproteinase; neutrophil collagenase; recombinant enzyme; article; catalysis; enzyme activity; enzyme purification; human; human cell; neutrophil; pH; pKa; temperature dependence; thermodynamics; Amino Acid Sequence; Biophysics; Comparative Study; Enzyme Stability; Fluorescent Dyes; Gelatinase A; Gelatinase B; Human; Hydrogen-Ion Concentration; In Vitro; Kinetics; Neutrophil Collagenase; Oligopeptides; Protons; Recombinant Proteins; Substrate Specificity; Support, Non-U.S. Gov't; Temperature; Thermodynamics
Fasciglione G.F., M.S. (2000). pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B. BIOPHYSICAL JOURNAL, 79(4), 2138-2149.
Fasciglione, G; Marini, S; D'Alessio, S; Politi, V; Coletta, M
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/53091
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