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Leucine Aminopeptidase: Bestatin Inhibition and a Model for Enzyme-Catalyzed Peptide Hydrolysis (X-Ray Crystallofraphy/Zinc Enzyme/Exopeptidase) STEPHEN K

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Leucine Aminopeptidase: Bestatin Inhibition and a Model for Enzyme-Catalyzed Peptide Hydrolysis (X-Ray Crystallofraphy/Zinc Enzyme/Exopeptidase) STEPHEN K Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6916-6920, August 1991 Biochemistry Leucine aminopeptidase: Bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis (x-ray crystallofraphy/zinc enzyme/exopeptidase) STEPHEN K. BURLEY*tt, PETER R. DAVID*§, AND WILLIAM N. LIPSCOMB* *Gibbs Chemical Laboratory, Harvard University, Cambridge, MA 02138; and tDepartment of Medicine, Brigham and Women's Hospital, Boston, MA 02115 Contributed by William N. Lipscomb, April 24, 1991 ABSTRACT The three-dimensional structures of native (11). In this paper, we present a detailed analysis of the bovine lens leucine aminopeptidase (EC 3.4.11.1) and its mechanism of inhibition of the enzyme by bestatin and complex with bestatin, a slow-binding inhibitor, have been discuss features of a mechanism of catalysis of peptide solved and exhaustively refined. The mode of binding of hydrolysis by LAP. This mechanism is in no way definitive, bestatin to leucine aminopeptidase may be similar to that of a but rather is intended to stimulate experimental studies ofthe tetrahedral intermediate that is thought to form during peptide biochemistry, including site-directed mutagenesis, of the bond hydrolysis. Bestatin binds in the active site with its LAPs. a-amino group and hydroxyl group coordinated to the zinc ion located in the readily exchangeable divalent cation binding site. Requirements for LAP-Catalyzed Peptide Hydrolysis Its phenylalanyl side chain is stabilized by van der Waals interactions with Met-270, Thr-359, Gly-362, Aa-451, and Hydrolytic cleavage of the amino-terminal peptide bond of a Met-454, which appear to form a terminal hydrophobic pocket. polypeptide substrate by LAP involves nucleophilic attack at The leucyl side chain binds in another hydrophobic cleft lined the carbonyl carbon atom and electrophilic attack at the by Asn-330, Ala-333, and fle-421. Hydrogen bonds involving amide NH of this bond. Candidates for nucleophilic attack active site residues Lys-262, Asp-273, Gly-360, and Leu-362 are could include a neutral or negatively charged side chain or an responsible for stabilizing the backbone nitrogen and oxygen attack by H20 or incipient OH- promoted by a positive atoms of bestatin. The mode of bestatin inhibition of leucine charge (e.g., Zn2 ). Candidates for subsequent proton dona- aminopeptidase is discussed and correlated with biochemical tion to permit bond cleavage could include H20 or a nearby studies of bestatin analogues. In addition, features of a mech- amino acid side chain with an appropriate pKa. Productive anism of catalysis of peptide hydrolysis by leucine aminopep- binding of a rapidly cleavable substrate would require the tidase are discussed. following events, not necessarily in order: (i) recognition of the amino-terminal group as NH+ or NH2 [the pKa of a Leucine aminopeptidase [LAP; cytosol aminopeptidase, polypeptide amino-terminal a-ammonium- group ordinarily a-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1] is a ranges from 7.7 to 8.3 (12)]; (it) binding ofthe P1 L-amino acid widely distributed cytosolic exopeptidase- which catalyzes side chain in an S, subsite that cannot productively bind the hydrolysis of amino acids from the amino terminus of lysine or arginine; (i) binding of the Pi L-amino acid side polypeptide chains (for reviews, see refs. 1-3). As their name chain in an S' subsite that cannot productively bind lysine, implies, the LAPs cleave leucyl substrates, although sub- arginine, proline, or hydroxyproline; and (iv) binding of the stantial rates of enzymatic cleavage are seen with most P' amino acid without preference as to either polarity or amino-terminal amino acids. However, residues P1 or PI chirality. should not be lysine or arginine, P1 should not be a D-amino acid, and P' should not be hydroxyproline or proline, using Deductions from LAP Crystallography the convention of Schechter and Berger (4). The amino acid occurring at P' does not seem to influence the rate ofcatalysis The structure of the active site of native LAP is illustrated in significantly (5). LAP also catalyzes the hydrolysis of amino Fig. 1A. Two zinc ions, separated by about 2.9 A, lie in the acid amides, alkylamides, arylamides, and hydrazides and floor ofthe active site, in chemical environments that are not has some esterase activity. equivalent. Results of the crystallographic refinement, along Bovine lens LAP is a hexameric enzyme of molecular with other biochemical data, identify which of the two sites weight 324,000, which consists of six identical subunits (6) of is more likely to exchange with divalent cations (7, 13). The molecular weight 54,000 and 12 zinc ions (7). A potent, zinc ion appearing in the top of Fig. IA is coordinated near slow-binding inhibitor of LAP, bestatin or [(2S,3R)-3-amino- the other zinc ion by the carboxylate oxygen atoms of 2-hydroxy-4-phenylbutanoyl]-L-leucine hydroxide, was iso- Asp-273 and Glu-334 and the side chain amino group of lated from culture filtrates of Streptomyces olivoreticuli (8) Lys-250 and has a refined temperature factor ofnearly 40 A2. and was shown to have a Ki = 20 nM for bovine lens LAP (9). The lower zinc ion is coordinated near the other zinc ion by The three-dimensional structure of the bestatin-inhibited the carboxylate oxygen atoms of Asp-255, Glu-334, and enzyme has been solved in our laboratory at 3-A resolution Asp-332 and by the carbonyl oxygen atom ofAsp-332 and has by x-ray crystallography using the multiple isomorphous Both the chemical replacement method with phase combination and density a refined temperature factor of 12 A2. modification (10). Recently, we reported the refinement of environments and the refined temperature factors suggest the structures of both the native enzyme and the LAP- bestatin complex at 2.32- and 2.25-A resolution, respectively Abbreviation: LAP, leucine aminopeptidase. tPresent address: Laboratory of Molecular Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, The publication costs of this article were defrayed in part by page charge NY 10021-6399. payment. This article must therefore be hereby marked "advertisement" §Present address: Department of Cell Biology, Stanford University in accordance with 18 U.S.C. §1734 solely to indicate this fact. School of Medicine, Stanford, CA 94305. 6916 Downloaded by guest on September 26, 2021 Biochemistry: Burley et A Proc. Natl. Acad. Sci. USA 88 (1991) 6917 A j~2k~\ 4 34 x % 7 -KQ -1 2 V2 2 B 50 4 FIG. 1. Stereo drawings of the enzyme active site. Residues involved in zinc ion binding are Lys-250, Asp-255, Asp-273, Asp-332, and Glu-334. The readily exchangeable zinc appears uppermost, and the two views are nearly identical. (A) Native (unligated) enzyme. (B) Enzyme-bestatin complex overlaid on the native enzyme active site. The bonds corresponding to the native enzyme are shown with broken lines. that it is the upper ofthe two zinc ions that readily exchanges 262 makes a salt bridge with the carboxylate oxygen atom of with other divalent cations in solution (see Fig. lA). Asp-332 that is not coordinating a zinc ion. Arg-336 appears Biochemical support for this identification is found in the to adopt two distinct conformations, one of which is close to work of Ricci et al. (14), who used nuclear magnetic reso- the two zinc ions. There is no evidence in the electron density nance spectroscopy to establish the proximity of a manga- map of a zinc-ligated water or a zinc-ligated hydroxide anion nese ion in the so-called activation site with the carbonyl in the vicinity of the readily exchangeable zinc ion, but a oxygen atom of N-(leucyl)-o-aminobenzenesulfonate when ligand may well be impossible to visualize at this resolution the enzyme inhibitor complex is formed in solution. The partly because of the apparent mobility of this zinc ion, as structure of the active site of the LAP-bestatin complex judged by its high temperature factor. Approximately 2.9 A described below reveals that the hydroxyl group of bestatin, from the more tightly bound zinc ion, an electron density which is analogous to the carbonyl oxygen atom of feature consistent with a weakly bound water molecule is N-(leucyl)-o-aminobenzenesulfonate, is close to the zinc visible in the electron density difference maps. Alternatively, binding site identified as the so-called activation site. We this feature may simply represent a noise peak. prefer the term readily exchangeable site to activation site The active site of the LAP-bestatin complex was found to because the degree of enzyme activation following metal contain electron density that could be clearly identified as substitution depends on the substrate used in the assay. For bestatin, which appeared to be present at full occupancy (see example, there is significant activation of LAP by Mg2' or Fig. 1B for a stereo drawing of the active site of the enzyme- Mn2+ when assayed using Leu-NH2 (7) but at most only bestatin complex). Binding of bestatin in the enzyme active 2-fold activation under the same conditions when assayed site does not significantly perturb the structure of the en- using Leu-Gly-NH(CH2)2-dansyl (13). zyme, even in the vicinity of the ligand (see Fig. 1B for the The native enzyme active site also contains two positively superposition of the two active site structures). Interactions charged amino acid side chains, Lys-262 and Arg-336. Lys- between the protein and the two zinc ions are preserved, and Downloaded by guest on September 26, 2021 6918 Biochemistry: Burley et al. Proc. Natl. Acad. Sci. USA 88 (1991) local structural changes are restricted to subtle shifts of the by van der Waals interactions with Met-270, Thr-359, Gly- active site residues that permit the two zinc ions to move 362, Ala-451, and Met-454, which appear to form a terminal slightly further apart (the two zinc ions are now equally well hydrophobic pocket.
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