Matrix Metalloproteinase Triple-Helical Peptidase Activities Are Differentially Regulated by Substrate Stability

Abstract

Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological destruction of tissues. The turnover of the collagen triple-helical structure has been ascribed to several members of the MMP family, but the determinants for collagenolytic specificity have not been identified. The present study has compared the triple-helical peptidase activities of MMP-1 and MMP-14 (membrane-type 1 MMP; MT1-MMP). The ability of each enzyme to efficiently hydrolyze the triple helix was quantified using chemically synthesized fluorogenic triple-helical substrates that, via addition of N-terminal alkyl chains, differ in their thermal stabilities. One series of substrates was modeled after a collagenolytic MMP consensus cleavage site from types I-III collagen, while the other series had a single substitution in the P(1)' subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P(1)' subsite was greatly favored by MMP-14 but disfavored by MMP-1. An increase in substrate triple-helical thermal stability led to the decreased ability of the enzyme to cleave such substrates, but with a much more pronounced effect for MMP-1. Increased thermal stability was detrimental to enzyme turnover of substrate (k(cat)), but not binding (K(M)). Activation energies were considerably lower for MMP-14 hydrolysis of triple-helical substrates compared with MMP-1. Overall, MMP-1 was found to be less efficient at processing triple-helical structures than MMP-14. These results demonstrate that collagenolytic MMPs have subtle differences in their abilities to hydrolyze triple helices and may explain the relative collagen specificity of MMP-1.

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

“…The hydrolysis of fTHP-15 by each of these MMPs has been reported previously (8,17,18,35,45) and occurs at the GlyϳLeu bond. Thus, fTHP-15 served as a well established point of reference.…”

“…These melting temperatures indicate fTHP thermal stabilities suitable for MMP kinetic analyses. The three fTHPs had similar stabilities, an important consideration as prior studies have shown that MMP hydrolytic activity toward both THPs and collagens can vary greatly due to differences in substrate thermal stability (35,41). The close similarity of thermal stability among the three fTHPs warranted our continued analysis of the effects of sequence variations.…”

“…All fTHPs were based on a consensus sequence derived from the collagenolytic MMP cleavage sites in human type I-III collagens (35). fTHP-15, fTHP-16, and fTHP-17 (see Table 1 for sequences) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase chemistry as described previously (8,17,18,30).…”

Enzymatic turnover of macromolecules generates long-lasting protein–water-coupled motions beyond reaction steady state

“…We used the collagen model substrate [Gly-Pro-4-hydroxyproline (Hyp)] 5 -Gly-Pro-Lys(7-methoxycoumarin-4-yl)-Gly-Pro-Gln-GlyCys(4-methoxybenzyl)-Arg-Gly-Gln-Lys(2,4-dinitrophenyl)-Gly-ValArg-(Gly-Pro-Hyp) 5 -NH 2 as an optimized recognition sequence for MT1-MMP (26,27). This substrate is biologically active and exhibits self-assembly capability to form triple-helical collagen-like monomers from its single-chain cognate peptide (13.5 kDa).…”

“…The FRET peptide (Gly-ProHyp) 5 -Gly-Pro-Lys(7-methoxycoumarin-4-yl)-Gly-Pro-Gln-Gly-Cys(4-methoxybenzyl)-Arg-Gly-Gln-Lys(2,4-dinitrophenyl)-Gly-Val-Arg-(Gly-Pro-Hyp) 5 -NH 2 was synthesized as described previously (26). Nonfluorogenic peptide substrates (for XAS experiments) were synthesized by Genscript.…”