Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, 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 A, respectively. Leucinal binds as a hydrated gem-diol to the active site of b1LAP), resembling the presumed gem-diolated 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 dizinc center and by interaction with Lys-262 and Arg-336. In the 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 b1LAP-leucinal structure, an unusually short hydrogen bond is found between two active site water molecules.
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Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138, USA.
Lipscomb
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D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007908:Lens, Crystalline / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000378:metabolism*; D014867:Water / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D011480:Protease Inhibitors; D014867:Water; C035766:leucinal; D007931:Leucyl Aminopeptidase; D007930:Leucine; D015032:Zinc