UNDERSTANDING THE P-1' SPECIFICITY OF THE MATRIX METALLOPROTEINASES -EFFECT OF S-1' POCKET MUTATIONS IN MATRILYSIN AND STROMELYSIN-1

Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P-1' position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of t hese enzymes show that their respective S-1' subsites differ primarily due to the amino acids present at positions 214 and 215. To examine t he role that these residues play in determining P-1' specificity, the amino acids at these positions in matrilysin have been replaced by tho se found in stromelysin (MAT:Y214L, MAT:A215V, and MAT:Y214L/A215V). T he specificity and activity of MAT:A215V are similar to those of wild type matrilysin. Both MAT:Y214L and MAT:Y214L/A215V, however, have P-1 ' 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 P-1' relative to wild type matrilysin. This is predominantly the result of an approxima te 5-fold decrease in k(cat). The K-M values only partially increase t oward the value observed for stromelysin. Studies of the pre-steady-st ate reaction of wild type and mutant matrilysin with substrates with L eu and Tyr residues in the P-1' position confirm that the K-M values f or these reactions reflect K-D values for substrate binding. Thus, rep lacement of a single tyrosine residue in the S-1' pocket of matrilysin by leucine alters its P-1' specificity to resemble that of stromelysi n. In contrast, alteration of the S-1' subsite of stromelysin (HFS:L21 4Y/V215A) to resemble matrilysin increases activity (i.e., higher k(ca t)/K-M) toward peptide substrates with both leucine and residues with aromatic side chains in the P-1' 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.