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PDBsum entry 1txr
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Spectroscopic and X-Ray crystallographic characterization of bestatin bound to the aminopeptidase from aeromonas (vibrio) proteolytica.
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Authors
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C.C.Stamper,
D.L.Bienvenue,
B.Bennett,
D.Ringe,
G.A.Petsko,
R.C.Holz.
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Ref.
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Biochemistry, 2004,
43,
9620-9628.
[DOI no: ]
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PubMed id
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Abstract
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Binding of the competitive, slow-binding inhibitor bestatin
([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from
Aeromonas proteolytica (AAP) was examined by both spectroscopic and
crystallographic methods. Electronic absorption spectra of the catalytically
competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the
presence of bestatin revealed that both of the divalent metal ions in AAP are
involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum
of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or
parallel-mode signal. These data indicate that the two Co(II) ions in AAP are
antiferromagnetically coupled yielding an S = 0 ground state and suggest that a
single oxygen atom bridges between the two divalent metal ions. The EPR data
obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with
both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex
was solved to 2.0 A resolution. Both side chains of bestatin occupy a
well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active
site. The amino acid residues ligated to the dizinc(II) cluster in AAP are
identical to those in the native structure with only minor perturbations in bond
length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in
the active site, displacing the bridging water molecule observed in the native
[ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex
and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1
and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl
oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In
addition, the NH(2) group of bestatin, which mimics the N-terminal amine group
of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A
combination of the spectroscopic and X-ray crystallographic data presented
herein with the previously reported mechanistic data for AAP has provided
additional insight into the substrate-binding step of peptide hydrolysis as well
as insight into important small molecule features for inhibitor design.
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