Structure determination and refinement of bovine lens leucine aminopeptidase and its complex with bestatin

• Published in: Journal of molecular biology Vol. 224; no. 1; pp. 113 - 140
• Main Authors: Burley, Stephen K., David, Peter R., Sweet, Robert M., Taylor, Allen, Lipscomb, William N.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 05.03.1992; Elsevier
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; bestatin; Binding Sites; Biological and medical sciences; Cattle; complex; crystal structure; cytosol aminopeptidase; Enzyme Stability; Enzymes and enzyme inhibitors; exopeptidase; eye lens; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Hydrolases; Lens, Crystalline - enzymology; Leucine - analogs & derivatives; Leucine - chemistry; Leucine - metabolism; leucine aminopeptidase; Leucyl Aminopeptidase - antagonists & inhibitors; Leucyl Aminopeptidase - chemistry; Leucyl Aminopeptidase - metabolism; Models, Molecular; Molecular Sequence Data; multiple isomorphous replacement; Protein Conformation; Sequence Alignment; X-ray crystallography; X-Ray Diffraction; X-ray crystallography; multiple isomorphous replacement; leucine aminopeptidase; bestatin; exopeptidase; Vertebrata; Mammalia; Bovine; Enzyme; Molecular complex; Lens; Artiodactyla; Crystallography; X ray diffraction; Spatial structure; Ungulata; Aminopeptidase (cytosol)
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(92)90580-D

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3·0 Å resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, this structure and the structure of the isomorphous native enzyme have been refined at 2·25 and 2·32 Å resolution, respectively, with crystallographic R-factors of 0·180 and 0·159, respectively. The current structural model for the enzyme includes the two zinc ions and 481 of the 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry, and is triangular in shape with a triangle edge length of 115 Å and maximal thickness of 90 Å. Monomers are crystallographically equivalent. Each is folded into two unequal α β domains connected by an α-helix to give a comma-like shape with approximate maximal dimensions of 90 Å × 55 Å × 55 Å. The secondary structural composition is 35% α-helix and 23% β-strand. The N-terminal domain (160 amino acid residues) mediates trimer-trimer interactions and does not appear to participate directly in catalysis, while the C-terminal domain (327 amino acid residues) is responsible for catalysis and binds the two zinc ions, which are less than 3 Å apart. These two metal ions are located near the edge of an eightstranded, saddle-shaped β-sheet. The zinc ion that has the lower temperature factor is co-ordinated by one carboxylate oxygen atom from each of Asp255, Asp332 and Glu334, and the carbonyl oxygen of Asp332. The other zinc ion, presumed to be readily exchangeable, is co-ordinated by one carboxylate oxygen atom of each of Asp273 and Glu334 and the side-chain amino group of Lys250. The active site also contains two positively charged residues, Lys262 and Arg336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 Å and thickness 10 Å. Access to this cavity is provided by solvent channels that run along the 2-fold symmetry axes. Bestatin binds to one of the active site zinc ions, and its phenylalanine and leucine side-chains occupy hydrophobic pockets adjacent to the active site. Finally, the relationship between bovine lens leucine aminopeptidase and tie homologous enzyme pep A from Excherichia coli is discussed.