Understanding the P1‘ Specificity of the Matrix Metalloproteinases: Effect of S1‘ Pocket Mutations in Matrilysin and Stromelysin-1

Abstract

Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P₁‘ position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of these enzymes show that their respective S₁‘ subsites differ primarily due to the amino acids present at positions 214 and 215. To examine the role that these residues play in determining P₁‘ specificity, the amino acids at these positions in matrilysin have been replaced by those found in stromelysin (MAT:Y214L, MAT:A215V, and MAT:Y214L/A215V). The specificity and activity of MAT:A215V are similar to those of wild type matrilysin. Both MAT:Y214L and MAT:Y214L/A215V, however, have P₁‘ specificities that are more similar to stromelysin than matrilysin. Specifically, these enzymes exhibit an 8- to 9-fold reduction in k_cat/K_M toward a peptide substrate with Leu in subsite P₁‘ relative to wild type matrilysin. This is predominantly the result of an approximate 5-fold decrease in k_cat. The K_M values only partially increase toward the value observed for stromelysin. Studies of the pre-steady-state reaction of wild type and mutant matrilysin with substrates with Leu and Tyr residues in the P₁‘ position confirm that the K_M values for these reactions reflect K_D values for substrate binding. Thus, replacement of a single tyrosine residue in the S₁‘ pocket of matrilysin by leucine alters its P₁‘ specificity to resemble that of stromelysin. In contrast, alteration of the S₁‘ subsite of stromelysin (HFS:L214Y/V215A) to resemble matrilysin increases activity (i.e., higher k_cat/K_M) toward peptide substrates with both leucine and residues with aromatic side chains in the P₁‘ position with only a partial increase in specificity for Leu. These increases in activity are the result of decreases in the K_M values for these reactions.