title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University,777 Glades Road, Boca Raton, Florida 33431-0991, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Lauer-Fields
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D024061:Collagen Type III / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D020689:Exopeptidases / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D024061:Collagen Type III; D005456:Fluorescent Dyes; D020689:Exopeptidases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases