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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 A 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 A
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 A and maximal thickness
of 90 A. Monomers are crystallographically equivalent. 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 A x 55 A x 55 A. The secondary
structural composition is 35% alpha-helix and 23% beta-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 A apart. These two metal ions are located near the
edge of an eight-stranded, saddle-shaped beta-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 A and
thickness 10 A. 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 the homologous enzyme pepA from Escherichia coli is
discussed.
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