Transition state analogue L-leucinephosphonic acid bound to bovine lens leucine aminopeptidase: X-ray structure at 1.65 A resolution in a new crystal form.
The three-dimensional structure of bovine lens leucine aminopeptidase (blLAP) complexed with L-Leucinephosphonic acid (LeuP) has been determined by molecular replacement using the structure of native blLAP as a starting model. Cocrystallization of the enzyme with the inhibitor yielded a new crystal form of space group P321 which has cell dimensions a = 130.4 A and c = 125.4 A. Refinement of the model against data from 7.0 to 1.65 A resolution resulted in a final structure with a crystallographic residual of 0.160 (R(free) = 0.191). The N-terminal amino group of LeuP is coordinated to Zn-489, one phosphoryl oxygen atom bridges both metal ions, and another phosphoryl oxygen atom is coordinated to Zn-488. The side chain of Arg-336 interacts with the inhibitor via three water molecules. LeuP resembles the presumed tetrahedral gem-diolate transition state after direct attack of a water or hydroxide ion nucleophile on the scissile peptide bond. On the basis of the LeuP binding mode and the previous structural and biochemical data, three plausible reaction pathways are evaluated. The two-metal ion mechanisms discussed herein share as common features a metal-bound hydroxide ion nucleophile and polarization of the carbonyl group by the zinc ions. Possible catalytic roles of Arg-336 and Lys-262 in the direct or indirect (through H2O) protonation of the leaving group, in the stabilization of a zinc-bound OH- nucleophile and in the stabilization of the negatively charged intermediate, are discussed. A site 3 metal ion approximately 12 A away from the active site 2 zinc ion probably serves a structural role.
Sträter
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Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138, USA.
Lipscomb
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D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides
D000900:Anti-Bacterial Agents; D063065:Organophosphonates; D010455:Peptides; C094802:leucine phosphonic acid; C016977:amastatin; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex