Abstract
Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.
Highlights
Triple-helical structures are resistant to most proteases
matrix metalloproteinase (MMP)-1, MMP-8, MMP-13, and MMP-14/ membrane-type 1 MMP (MT1-MMP) are collagenases that hydrolyze the triple helix of fibrillar type I–III collagens with varying efficiencies at a single GlyϳIle/Leu site
Three fluorogenic triple-helical peptides (fTHPs) were utilized in the present study (Fig. 1 and Table 1). fTHP-15 incorporates a consensus sequence from type I–III collagens (Table 2) and provides for convenient monitoring of triple-helical peptidase activity by collagenolytic MMP family members
Summary
Triple-helical structures are resistant to most proteases. several members of the matrix metalloproteinase (MMP) subfamily of zinc-dependent endopeptidases catalyze the hydrolysis of triple helices [5]. An alternative and novel approach is to examine how MMPs interact with customized triple-helical peptides (THPs) through secondary binding sites (exosites)/allosteric sites [28]. We recently have found that by varying the composition of collagen model sequences within THPs we can explore unique binding interactions in the CAT and HPX domains of MMP-1 [18]. In the present study, we hypothesized that probing the CAT and HPX domains of collagenolytic MMPs via THPs with varying contents of collagen model sequences will reveal different binding site interactions. To test this hypothesis, we utilized three fluorogenic THP substrates (Table 1). Overall activities and individual kinetic parameters were quantified for MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14 catalysis of each substrate
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