Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.35 (matrix metalloproteinase 9)
 2,207 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The peptide substrate specificities of two matrix metalloproteinases (MMPs), interstitial collagenase (MMP-1), and 92-kDa gelatinase (MMP-9), have been examined. Starting with the parent substrate, Dnp-Pro-Leu-Gly approximately Leu-Trp-Ala-D-Arg-NH2, four separate substrate mixtures were synthesized at subsites P2(Leu) through P2'(Trp). These mixtures contained either naturally occurring L-amino acids, D-amino acids, or either of two distinct sets of miscellaneous amino acids. Combined, these mixtures gave 88 unique substitutions at each position and, over the four subsites, represented 352 potential substrates. Optimal substrates were identified using a combined high performance liquid chromatography/mass spectrometry analysis as previously reported. The results gave an extended profile of the substrate specificities for both MMP-1 and MMP-9 at subsites P2(Leu) through P2'(Trp). Using the data obtained from the mapping, a new peptide substrate, Dnp-Pro-Cha-Abu approximately Smc-His-Ala-D-Arg-NH2 (where Dnp is 2,4-dinitrophenyl, Cha is cyclohexylalanine, Abu is alpha-aminobutyric acid, and Smc is S-methylcysteine) was designed and characterized. This peptide showed a 36-fold improvement in turnover (kcat/Km) versus the parent substrate by interstitial collagenase. In addition, some collagenase subsite specificities described here were found to be different from those previously reported. Experimental data show that the observed selectivity is dependent on the original peptide template employed, which has broader implications for substrate specificity studies.
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PMID:Characterization of the peptide substrate specificities of interstitial collagenase and 92-kDa gelatinase. Implications for substrate optimization. 780 5

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.
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PMID:Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. 862 38

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.
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PMID:Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. 1090 30

During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin-8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high-performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O-linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post-translational modifications at P2' and P5', respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T-cell activation. These data lend support to the thesis that extracellular proteolysis and other post-translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes.
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PMID:Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis. 1187 87