Molecular structure of leucine aminopeptidase at 2.7-A resolution.

Abstract

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-.ANG. resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-.ANG. resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 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 .ANG. and maximal thickness of 90 .ANG.. The monomers are crystallographically equivalent and each is folded into two unequal .alpha./.beta. domains connected by an .alpha.-helix to give a comma-like shape with approximate maximal dimensions of 90 .times. 55 .times. 55 .ANG.3. The secondary structural composition is 40% .alpha.-helix and 19% .beta.-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interaction and does not appear to participate directly in catalysis and binds the two zinc ions, which are 2.88 .ANG. apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped .beta.-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-225, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 .ANG. and thickness 10 .ANG.. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.