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.

Neutrophil collagenase MMP-81 is a Zn 2ϩ metallo-endopeptidase, which is able to cleave native triple-helical collagen I (1-3) at a specific peptide bond between Gly 775 and the residue at position 776 (which can be either Leu or Ile), leading to the formation of one-quarter and three-quarter fragments (4). MMP-8 is predominantly expressed by neutrophil precursors (5, 6), but very recently it has been demonstrated that it is also expressed in human articular chondrocytes (7) and that it might also be responsible for aggrecanase activity (8, 9). MMP-8 is expressed initially as a proenzyme and stored in the specific granules of neutrophils (10). The activation of MMP-8 can be accomplished either by mercurials, oxidative processes, and proteinases (11-14) thus removing a cysteinyl residue, which coordinates the Zn 2ϩ atom, rendering the enzyme inactive. In particular, it was observed that cathepsin G, a neutrophil serine proteinase, and stromelysin MMP-3 activate proMMP-8 through two different processes, the first one cleaving the Phe 79 -Met 80 peptide bond (11), whereas MMP-3 activates through the cleavage of the Gly 78 -Phe 79 peptide bond (15). Therefore, the resulting active MMP-8 displays two different N termini (i.e. Met 80 if activated by cathepsin G and Phe 79 if activated by MMP-3), and it has been shown that these two forms display meaningful differences in the catalytic activity toward synthetic substrates or inhibitors (16,17). The final active enzyme is made of a catalytic domain, where the Zn 2ϩ atom coordinated to 3 histidyl residues is located, and a hemopexin-like domain, which is connected to the catalytic domain through a linker region (18,19).Collagen I is one of the major components of the extracellular matrix for most tissues, such as skin, tendon, blood vessels, cartilage, bones, and basal laminae (20). It displays a triplehelical structural arrangement (21), which is kept by most of the molecule during the first cleavage event by MMP-8 (22), even though three-dimensional structures of collagenases indicate that the substrate binding cleft appears too narrow to accommodate a triple-helical collagen molecule. The two sets of data are not necessarily in contradiction, because a partial unwinding of collagen may be envisaged during the enzymatic action (23-25) while keeping the overall three-dimensional structure of the whole collagen molecule.Several studies have shown that, although the actual enzymatic action of collagenases occurs in the catalytic domain, the hemopexin-like domain plays an important role, because its * This work was supported in part by funds from the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST COFIN MM03185591 to M. C.) and the Deutsche Forschungsgemeinschaft, Bonn, Grants SFB 549 and SFB A5 (to H.T.) and Project Ts 8/35 (to H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely ...

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.

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The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation.The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.Enzyme. Ascorbate oxidase