Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn(++)-endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in specific developmental or pathological conditions, including human periodontal diseases.

Matrix Metalloproteinases: A Review

Zinc coordination, function, and structure of zinc enzymes and other proteins

The matrix-degrading metalloproteinases are an intriguing family of enzymes that have evolved to digest specific extracellular matrix components. The expression of these enzymes is very highly regulated and can be controlled transcriptionally by a number of growth factors, tumor promoters, oncogenes, and hormones. It is suggested that the coordinated regulation of matrix metalloproteinases and their inhibitors by these agents modify the integrity of the extracellular matrix. These modifications may, at least in part, be responsible for mediating the effects of these factors on complex physiological processes.

The matrix-degrading metalloproteinases.

Metalloporphyrins are widely used throughout the biosphere and of these heme (iron protoporphyrin IX, Fig. 1) is one of the most abundant and widely used. Heme shuttles electrons between proteins as in mitochondrial respiration or transports and stores O2 as with the globins. The role of heme in more active enzymatic chemical transformation began to be appreciated just after the discovery by Mason1 and Hayaishi2 that O2 O atoms can be enzymatically incorporated into organic substrates which represented the seminal discovery of oxygenases. While the enzymes used in these studies did not contain heme, it was not too long before heme-containing oxygenases also were discovered. In 1958 Klingenberg3 and Garfinkel4 found an unusual pigment in microsomes that when reduced in the presence of CO generated a spectrum with a peak at 450 nm instead of the expected 420 nm peak. Hence the name P450 was born. In 1964 Omura and Sato5,6 showed that this “pigment” is actually a protein and the function of this strange heme protein became clear in a seminal study by Estabrook et al.7 that demonstrated the involvement of the 450 nm pigment in steroid hydroxylation. Thus by the mid-1960s it was established that heme plays an active role in biology by somehow catalyzing the hydroxylation of organic substrates. While these discoveries certainly mark the beginning of modern approaches to studying heme enzyme oxygenases, the enzymatic role of heme dates much earlier to 1903 when horseradish peroxidase (HRP) was described.8 Indeed, owing to the ease of purification and stability of the various intermediates, HRP dominated heme enzyme studies until P450 was discovered.



Figure 1

Structure of iron protoporphyrin IX.



Heme enzymes can catalyze both reductive and oxidative chemistry but here we focus on those that catalyze oxidation reactions, and especially those for which crystal structures are available. There are two broad classes of heme enzyme oxidants: oxygenases that use O2 to oxidize, usually oxygenate, substrates and peroxidases that use H2O2 to oxidize, but not normally oxygenate, substrates. Of the two oxidants molecular oxygen is the most unusual because even though the oxidation of nearly all biological molecules by O2 is a thermodynamically favorable process, O2 is not a reactive molecule. The reason, of course, is that there is a large kinetic barrier to these reactions owing to O2 being a paramagnetic molecule so the reaction between a majority of biological molecules that have paired spins is a spin forbidden process. Overcoming this barrier is why Nature recruited transition metals and heme into enzyme active sites. As shown in Fig. 2, heme oxygenases bind O2 and store the O2 oxidizing equivalents in the iron, porphyrin, and/or amino acid side chains for further selective oxidation of substrates. Peroxidases use H2O2 as the oxidant and while not having the O2 spin barrier, H2O2 presents its own problems. The reaction between H2O2 and transition metals generates toxic hydroxyl radicals in the well known Fenton chemistry9 which would be highly destructive to enzyme active sites. As illustrated in Fig. 2, all heme oxidases are at some point in the catalytic cycle peroxidases. Molecular oxygen must first be reduced by two electrons to the peroxide level before the interesting chemistry starts: cleavage of the O-O bond. This bond can cleave either homolytically, which gives two hydroxyl radicals, or heterolytically to effectively give H2O and a naked O atom with only 6 valence electrons. Since the release of hydroxyl radicals in the active site must, in most cases, be avoided Nature has engineered heme enzyme active sites to ensure that the heterolytic pathway dominates.



Figure 2

Oxygen and peroxide activation by heme enzymes. Oxygenases like P450 must have the iron reduced to ferrous (Fe(II) or Fe2+) before O2 can bind. The oxy complex is best described as ferric-superoxide, Fe(III)-OO−. A second electron transfer results ...



The list of heme enzymes is substantial and thus it is necessary to be selective on which to discuss in detail. It may appear that a disproportionate amount of space is devoted to peroxidases and P450s. This is true and admittedly reflects the author’s own interests and area of expertise. Additionally, however, peroxidases are the most extensively studied heme enzymes and have provided fundamental insights into the chemistry and structure shared by many other enzymes. The other enzymes to be discussed were selected owing to both subtle variations on common themes and novel features that Nature selected for specific biological function.

Heme Enzyme Structure and Function

Design and therapeutic application of matrix metalloproteinase inhibitors.

The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagenase), and putative metalloproteinase (PUMP or matrilysin) have been examined by measuring the rate of hydrolysis of over 50 synthetic oligopeptides covering the P4 through P4' subsites of the substrate. The peptides investigated in this paper were those employed in our previous study which systematically examined the sequence specificity of human fibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J. Biol. Chem. 266, 6747]. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured under first-order conditions ([S0] << KM), and kcat/KM values have been calculated from the initial rates. The specificities of these five metalloproteinases are similar, but distinct, with the largest differences occurring at subsites P1, P1', and P3'. The specificities of the two gelatinases are the most similar to each other. They tolerate only small amino acids such as Gly and Ala in subsite P1. In contrast, larger residues such as Met, Pro, Gln, and Glu are also accommodated well by PUMP. All five enzymes prefer hydrophobic, aliphatic residues in subsite P1'. PUMP exhibits a stronger preference for Leu in this subsite than is shown by the other enzymes. The P3' subsite specificities of the gelatinases and collagenases are very similar but different from those of PUMP which particularly prefers Met in this position. The specificity data from this study allow the design of optimized substrates and selective inhibitors for these metalloproteinases.

Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin)

Six collagenases present in the culture filtrate of Clostridium histolyticum have been purified to homogeneity. Chromatography over hydroxylapatite, Sephacryl S-200, and L-arginine-Affi-Gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester, and elastin. Reactive Red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among four fractions. The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex. All six collagenases, designated alpha, beta, gamma, delta, epsilon and zeta by the order of their purification, are highly active against collagen and devoid of other proteolytic activities. Each exhibits a single band on sodium dodecyl sulfate-polyacrylamide gels. Two distinct subspecies of the alpha and gamma enzymes have been isolated, which have the same molecular weight and activity but different isoelectric points. There is some less pronounced microheterogeneity for the other collagenases. On the basis of their activities toward native collagen and the synthetic peptide 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), the six collagenases are divided into two classes. Class I collagenases (alpha, beta, and gamma) have high collagenase activity and moderate FALGPA activity while the class II collagenases (sigma, epsilon, and sigma) have moderate collagenase and high FALGPA activities. The relationship between these six collagenases and other reported to have been isolated in the literature has also been examined.

Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography

The reaction of horseradish peroxidase (HRP) with H2O2 has been studied in 50% v/v methanol/water over the 25.0 to -36.0 degrees C temperature range by using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [H2O2] much greater than [HRP]. Arrhenius plots for the pseudo-first-order rate constant kobs were linear over the 17.6 to -36.0 degrees C temperature range studied with an activation energy of 4.8 +/- 0.5 kcal/mol. Above 0 degrees C, kobs varies linearly with peroxide concentration. However, saturation kinetics are observed below -16.0 degrees C, indicating that there is at least one reversible elementary step in this reaction. Double-reciprocal plots at -26.0 degrees C at pH* 7.3 for the reaction give kappa max(obs) = 163 s-1 and KM = 0.190 mM. Rapid-scan optical studies carried out at -35.0 degrees C with [H2O2] much greater than KM reveal the presence of a transient intermediate referred to as compound 0 whose conversion to compound I is rate limiting. The Soret region of the optical spectrum of compound 0 resembles that of a "hyperporphyrin" with prominent bands near 330 and 410 nm. The temperature dependencies of kappa max(obs) and KM have been measured over the -16.0 to -26.0 degrees C range and give an activation energy for kappa max(obs) of 1.6 +/- 0.7 kcal/mol and an enthalpy of formation for compound 0 of 4.0 +/- 0.7 kcal/mol.

Elementary steps in the formation of horseradish peroxidase compound I: direct observation of compound 0, a new intermediate with a hyperporphyrin spectrum.

A series of experiments has been carried out to characterize 58-kDa human neutrophil collagenase (HNC) and compare it with human fibroblast collagenase (HFC). N-Terminal sequencing of latent and spontaneously activated HNC shows that it is a distinct collagenase that is homologous to HFC and other members of the matrix metalloproteinase gene family. Activation occurs autolytically by hydrolysis of an M-L bond at a locus homologous to the Q80-F81-V82-L83 autolytic activation site of HFC. This releases a 16-residue propeptide believed to contain the "cysteine switch" residue required for latency. Polyclonal antibody raised against HNC cross-reacts with HFC but with none of the other major human matrix metalloproteinases examined. Treatment of HNC with endoglycosidase F or N-glycosidase F indicates that it is glycosylated at multiple sites. The deglycosylated latent and spontaneously activated enzymes have molecular weights of approximately 44K and 42K, respectively. Differences in the carbohydrate processing of HFC and HNC may determine why HFC is a secreted protein while HNC is stored in intracellular granules. The kinetic parameters kcat and KM for the hydrolysis of the interstitial collagen types I, II, and III in solution by both collagenases have been determined. The strong preferences of HNC for type I collagen and of HFC for type III collagen found in earlier studies have been confirmed. The preference of HNC for type I over type III collagen is almost abolished when fibrillar collagens are used as substrates, but the preference for HFC for type III over type I collagen is only partially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase.

The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen.

Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum.

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