Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the proces s of a variety of diseases such as arthritis, atherosclerosis, and tumor ce ll metastasis. To study the mechanisms of MMP action on collagenous substra tes, we have constructed homotrimeric triple-helical peptide (THP) models o f the collagenase cleavage sites in types I and II collagen. The THPs incor porate either the alpha 1(I)772-786 or the alpha 1(II)772-783 sequence. The alpha 1(I)772-786 and alpha 1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigati ons using the alpha 1(I)772-786 THP showed hydrolysis by MMP-2, MMP-13, and a COOH-terminal domain-deleted MMP-1 (MMP-1(Delta(243-450))) but not by MM P-3 or a COOH-terminal domain-deleted MMP-3 (MMP-3(Delta(248-460))). Kineti c analyses showed a k(cat)/K-m value of 1,808 s(-1) M-1 for MMP-1 hydrolysi s of alpha 1(I)772-786 THP, approximately 10-fold lower than for type I col lagen. The effect is caused primarily by relative K-m values. MMP-2 and MMP -13 cleaved the THP more rapidly than MMP-1, but MMP-2 cleavage occurred at distinct multiple sites. Comparison of MMP-1 and MMP-1(Delta(243-450)) hyd rolysis of alpha 1(I)772-786 THP showed that both can cleave a triple-helic al substrate with a slightly higher K-m value for MMP-1(Delta(243-450)). We propose that the COOH-terminal domain of MMPs is necessary for orienting w hole, native collagen molecules but may not be necessary for binding to and cleaving a THP, This proposal is consistent with the large distance betwee n the MMP-1 catalytic and COOH-terminal domains observed by three-dimension al structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity.