Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, nonfluorogenic and fluorogenic triplehelical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triplehelical peptides were assembled by either (a) covalent branching or (b) selfassociation driven by hydrophobic interactions. Fluorogenic triplehelical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5 positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP 1 ~ MMP-1(Δ243-450) ~ MMP-2 >> MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic singlestranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triplehelical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that triplehelical peptidase activity can be distinguished from collagenolytic activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.
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