Matrix metalloproteinase

The MMPs are initially synthesized as inactive zymogens with a pro-peptide domain that must be removed before the enzyme is active. The pro-peptide domain is part of the "cysteine switch." This contains a conserved cysteine residue that interacts with the zinc in the active site and prevents binding and cleavage of the substrate, keeping the enzyme in an inactive form. In the majority of the MMPs, the cysteine residue is in the conserved sequence PRCGxPD. Some MMPs have a prohormone convertase cleavage site (Furin-like) as part of this domain, which, when cleaved, activates the enzyme. MMP-23A and MMP-23B include a transmembrane segment in this domain.[9]

X-ray crystallographic structures of several MMP catalytic domains have shown that this domain is an oblate sphere measuring 35 x 30 x 30 Å (3.5 × 3 x 3 nm). The active site is a 20 Å (2 nm) groove that runs across the catalytic domain. In the part of the catalytic domain forming the active site there is a catalytically important Zn²⁺ ion, which is bound by three histidine residues found in the conserved sequence HExxHxxGxxH. Hence, this sequence is a zinc-binding motif.

The gelatinases, such as MMP-2, incorporate Fibronectin type II modules inserted immediately before in the zinc-binding motif in the catalytic domain.[10]

The catalytic domain is connected to the C-terminal domain by a flexible hinge or linker region. This is up to 75 amino acids long, and has no determinable structure.

The hemopexin-like C-terminal domain of MMP9 PDB 1itv

The C-terminal domain has structural similarities to the serum protein hemopexin. It has a four-bladed β-propeller structure. β-Propeller structures provide a large flat surface that is thought to be involved in protein-protein interactions. This determines substrate specificity and is the site for interaction with TIMP's (tissue inhibitor of metalloproteinases). The hemopexin-like domain is absent in MMP-7, MMP-23, MMP-26, and the plant and nematode. The membrane-bound MMPs (MT-MMPs) are anchored to the plasma membrane via a transmembrane or a GPI-anchoring domain.

There are three catalytic mechanisms published.

• In the first mechanism, Browner M.F. and colleagues[11] proposed the base-catalysis mechanism, carried out by the conserved glutamate residue and the Zn²⁺ ion.
• In the second mechanism, the Matthews-mechanism, Kester and Matthews[12] suggested an interaction between a water molecule and the Zn²⁺ ion during the acid-base catalysis.
• In the third mechanism, the Manzetti-mechanism, Manzetti Sergio and colleagues[13] provided evidence that a coordination between water and zinc during catalysis was unlikely, and suggested a third mechanism wherein a histidine from the HExxHxxGxxH-motif participates in catalysis by allowing the Zn²⁺ ion to assume a quasi-penta coordinated state, via its dissociation from it. In this state, the Zn²⁺ ion is coordinated with the two oxygen atoms from the catalytic glutamic acid, the substrate's carbonyl oxygen atom, and the two histidine residues, and can polarize the glutamic acid's oxygen atom, proximate the scissile bond, and induce it to act as reversible electron donor. This forms an oxyanion transition state. At this stage, a water molecule acts on the dissociated scissile bond and completes the hydrolyzation of the substrate.

Use of bioinformatic methods to compare the primary sequences of the MMPs suggest the following evolutionary groupings of the MMPs:

• MMP-19
• MMPs 11, 14, 15, 16, and 17
• MMP-2 and MMP-9
• All the other MMPs

Analysis of the catalytic domains in isolation suggests that the catalytic domains evolved further once the major groups had differentiated, as is also indicated by the substrate specificities of the enzymes.

The most commonly used groupings (by researchers in MMP biology) are based partly on historical assessment of the substrate specificity of the MMP and partly on the cellular localization of the MMP. These groups are the collagenases, the gelatinases, the stromelysins, and the membrane-type MMPs (MT-MMPs).

• The collagenases are capable of degrading triple-helical fibrillar collagens into distinctive 3/4 and 1/4 fragments. These collagens are the major components of bone, cartilage and dentin, and MMPs are the only known mammalian enzymes capable of degrading them. The collagenases are No. 1, No. 8, No. 13, and No. 18. In addition, No. 14 has also been shown to cleave fibrillar collagen, and there is evidence that No. 2 is capable of collagenolysis. In MeSH, the current list of collagenases includes No. 1, No. 2, No. 8, No. 9, and No. 13. Collagenase No. 14 is present in MeSH but not listed as a collagenase, while No. 18 is absent from MeSH.
• The main substrates of the gelatinases are type IV collagen and gelatin, and these enzymes are distinguished by the presence of an additional domain inserted into the catalytic domain. This gelatin-binding region is positioned immediately before the zinc-binding motif, and forms a separate folding unit that does not disrupt the structure of the catalytic domain. The gelatinases are No. 2 and No. 9.
• The stromelysins display a broad ability to cleave extracellular matrix proteins but are unable to cleave the triple-helical fibrillar collagens. The three canonical members of this group are No. 3, No. 10, and No. 11.
• All six membrane-type MMPs (No. 14, No. 15, No. 16, No. 17, No. 24, and No. 25) have a furin cleavage site in the pro-peptide, which is a feature also shared by No. 11.

However, it is becoming increasingly clear that these divisions are somewhat artificial as there are a number of MMPs that do not fit into any of the traditional groups.

Matrix metalloproteinases combines with the metal binding protein, metallothionine; thus helping in metal binding mechanism.

Doxycycline, at subantimicrobial doses, inhibits MMP activity, and has been used in various experimental systems for this purpose, such as for recalcitrant recurrent corneal erosions. It is used clinically for the treatment of periodontal disease and is the only MMP inhibitor that is widely available clinically. It is sold under the trade name Periostat by the company CollaGenex. Minocycline, another tetracycline antibiotic, has also been shown to inhibit MMP activity.

A number of rationally designed MMP inhibitors have shown some promise in the treatment of pathologies that MMPs are suspected to be involved in (see above). However, most of these, such as marimastat (BB-2516), a broad-spectrum MMP inhibitor, and cipemastat (Ro 32-3555), an MMP-1 selective inhibitor, have performed poorly in clinical trials. The failure of Marimastat was partially responsible for the folding of British Biotech, which developed it. The failure of these drugs has been due largely to toxicity (in particular, musculo-skeletal toxicity in the case of broad spectrum inhibitors) and failure to show expected results (in the case of trocade, promising results in rabbit arthritis models were not replicated in human trials). The reasons behind the largely disappointing clinical results of MMP inhibitors is unclear, especially in light of their activity in animal models.