A rationalization of the acidic pH dependence for stromelysin-1 (matrix metalloproteinase-3) catalysis and inhibition

Abstract

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His224. This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu202 and His224. The acidic pK(a) 5.6 is attributed to the Glu202·zinc·H2O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His224-dependent substructure is identified as a tripeptide (Pro221-Leu222-Tyr223) that forms the substrate cleft lower wall. Substrate binding induces a β- conformation in this sequence, which extends and anchors the larger β-sheet of the enzyme·substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this 'β-anchor' may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu202 and His224 allows the design of highly selective inhibitors.