2-METAL ION MECHANISM OF BOVINE LENS LEUCINE AMINOPEPTIDASE - ACTIVE-SITE SOLVENT STRUCTURE AND BINDING MODE OF L-LEUCINAL, A GEM-DIOLATE TRANSITION-STATE ANALOG, BY X-RAY CRYSTALLOGRAPHY

The three-dimensional structures of bovine lens leucine aminopeptidase (blLAP) complexed with L-leucinal and of the unliganded enzyme have been determined at crystallographic resolutions of 1.9 and 1.6 Angstrom, respectively. Leucinal binds as a hydrated gem-diol to the active site of blLAP, resembling the presumed gem-diolate intermediate in the catalytic pathway. One hydroxyl group bridges the two active site metal ions, and the other OH group is coordinated to Zn1. The high-resolution structure of the unliganded enzyme reveals one metal-bound water ligand, which is bridging both zinc ions. Together, these structures support a mechanism in which the bridging water ligand is the attacking hydroxide ion nucleophile. The gem-diolate intermediate is probably stabilized by four coordinating bonds to the di-zinc center and by interaction with Lys-262 and Arg-336. In this mechanism, Lys-262 polarizes the peptide carbonyl group, which is also coordinated to Zn1. The Arg-336 side chain interacts with the substrate and the gem-diolate intermediate via water molecules. Near Arg-336 in the blLAP-leucinal structure, an unusually short hydrogen bond is found between two active site water molecules.