MMP-12 catalytic domain recognizes triple helical peptide models of collagen V with exosites and high activity

Matrix metalloproteinase (MMP)-12 ( or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha 1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was sub-micromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K-m toward this substrate by decreasing K-m, just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha 1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha 1(V) THP protected MMP-12 from the NMR line broadening effects of Gd center dot EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha 1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha 1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.