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

• Published in: Biochemistry (Easton) Vol. 43; no. 36; pp. 11474 - 11481
• Main Authors: Minond, Dmitriy, Lauer-Fields, Janelle L, Nagase, Hideaki, Fields, Gregg B
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.09.2004
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Collagen Type I - metabolism; Collagen Type II - metabolism; Collagen Type III - metabolism; Enzyme Activation; Enzyme Stability; Exopeptidases - chemistry; Exopeptidases - metabolism; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - metabolism; Humans; Hydrolysis; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases - chemistry; Metalloendopeptidases - metabolism; Molecular Sequence Data; Protein Structure, Secondary; Substrate Specificity; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048938i

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 P1‘ subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P1‘ 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.