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

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, r esulting in a uniquely sharp acidic catalytic optimum, which has recently b een attributed to His(224). This suggests the presence of a critical, but u nidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from correspondi ng crystal structures, we isolated contributions to the pH dependence of ca talysis and inhibition of active site residues Glu(202) and His(224). The a cidic pK(a) 5.6 is attributed to the Glu(202). zinc . H2O complex, consiste nt with a role for the invariant active site Glu as a general base in MMP c atalysis, The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Su bstrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme . substrate complex and ap pears to be essential for productive substrate binding. Because the PXY tri peptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striki ng acidic profile of stromelysin-1 defined by the combined ionization of Gl u(202) and His(224) allows the design of highly selective inhibitors.