Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.
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PMID:Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins. 846 59

Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.
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PMID:Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase. 1201 Oct 42

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.
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PMID:Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities. 1529 30

The mechanism of triple helical collagen unwinding and cleavage by collagenases in the matrix metalloproteinase (MMP) family is complex and remains enigmatic. Recent reports show that triple helicase activity is initiated by the hemopexin C domain of membrane type 1-MMP, whereas catalytically inactive full-length interstitial collagenase (MMP-1) exhibits full triple helicase functionality pointing to active site determinants that are needed to complete the triple helicase mechanism. In MMP-8, the neutrophil collagenase, a conserved Gly at the S(3)' substrate specificity subsite is replaced by Asn(188) that forms a highly unusual cis bond with Tyr(189), a conserved active site residue in the collagenases. Only in MMP-1 is the S(3)' Gly also replaced, and there too a cis configured Glu-Tyr occurs. Thus, this high energy peptide bond coupled to the canonical Tyr may be important in the collagenolytic process. In a systematic mutagenesis investigation of the MMP-8 S(3)' subsite we found that introducing an S(3)' Gly(188) into MMP-8 reduced collagenolytic efficiency by approximately 30% with a corresponding reduction in cleavage of a synthetic peptide fluorescence resonance energy transfer substrate analogue of the alpha2(I) collagen chain cleavage site. The substitution of Asn(188) to Leu, a hydrophobic residue of similar size to the highly polar Asn and designed to retain the cis bond, revealed the importance of hydrogen bonding to bound substrate with both collagenolytic and peptidic activities reduced approximately 3-fold. In contrast, the specificity for type I collagen of the mutant Y189F dropped 3-fold without any significant alteration in general peptidase activity. Therefore, S(3)' and in particular the hydrogen bonding potential of Tyr(189) is a specific molecular determinant for MMP-8 triple helicase activity. The cis bond connection to Asn(188) juxtaposes these two side chains for closely spaced hydrogen bonding with substrate that improves collagenolytic and general catalytic efficiency that could be exploited for new collagenase-specific inhibitor drugs.
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PMID:Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond. 1553 38

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.
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PMID:The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity. 1706 55

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