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

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.

pH dependence of the enzymatic processing of collagen I by MMP-1 (fibroblast collagenase), MMP-2 (gelatinase A), and MMP-14 ectodomain

“…Since it is known that MMP-2 binds collagen I preferentially by the fibronectin-like domain [11,13], this difference might underlie the possibility that the a-1 chain interaction involves a different portion of the collagen binding domain of MMP-2 with respect to that binding the a-2 chain. In this respect, it is very important to underline that the pK U values for the a-1 chain are very similar to those reported for the interaction of MMP-2 with synthetic substrates [31]. Although a comparison between data on synthetic macromolecular substrates is not straightforward, since the enzyme-substrate interface is obviously different, this evidence suggests that in MMP-2 similar groups are possibly involved in the binding both to the synthetic substrate and to the a-1 chain of collagen I.…”

“…This behavior clearly suggests that binding of the three MMPs involves different topological regions of the collagen I triple helix and occurs through a different interaction mode for the two types of chains. In this respect, it is important to remark as for MMP-2 a close similarity is observed for the values of the three pK U with the a-1 chain of collagen I (see Table 1) and those obtained with synthetic substrates [31], indicating that these protonlinked groups belong unequivocally to the free enzyme and no contribution comes from the interacting substrate. On the other hand, the drastically different values of pK U between the synthetic substrates and the collagen chains observed for MMP-1 and ect-MMP-14 (see Table 1) clearly indicate that in these enzymes the mode of interaction with collagen chains is not regulated by the same residues involved in the reactivity modulation of the enzymatic activity toward synthetic substrates.…”

“…This obviously represents an additional difficulty for the quantitative analysis, since (unlike synthetic small substrates [31]) our observations are influenced by pH-dependent structural changes of both the enzyme(s) and the macromolecular substrate.…”

“…As an example, whereas in the bloodstream the pH is maintained around 7.4 ± 0.1, in the synovial liquid of joints the pH is much higher, being 8.1 ± 0.1 under normal conditions [26]; in both cases, inflammation (such as in osteoarthritis) brings about a lowering of the pH to 7.1 ± 0.1 and 7.6 ± 0.1, respectively [26]. Up to now, investigation of the pH dependence of enzymatic properties of MMPs has been carried out for synthetic substrates on porcine collagenases and gelatinases [27,28], on stromelysin MMP-3 [29,30], and on MMP-8, MMP-2, and MMP-9 [31], whereas for a macromolecular substrate an investigation of the pH dependence has been undertaken only for the enzymatic activity of MMP-8 toward collagen I [24]. In this paper we have extended the investigation of collagen I to the pH dependence of the enzymatic activity of MMP-1 (a collagenase), MMP-2 (a gelatinase), and the ectodomain of MMP-14 (ect-MMP-14; a membrane-type MMP).…”

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

“…From the analysis of the dependence on the substrate concentration at each pH value according to Eqs. (2) and (3) Figure 2A), a pH value quite low compared with other metallopeptidases (Fasciglione et al , 2000 ). Further, the pH dependence of K m ( Figure 2C) indeed suggests that the substrate affi nity tends to increase at pH lower than 7.5, as K m decreases.…”