Spectroscopic and X-ray Crystallographic Characterization of Spectroscopic and X-ray Crystallographic Characterization of Bestatin Bound to the Aminopeptidase from Bestatin Bound to the Aminopeptidase from Aeromonas (Vibrio) proteolytica

Binding of the competitive, slow-binding inhibitor bestatin ([(2 S ,3 R )-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 Å 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 molecul e observed in the native [ZnZn(AAP)] structure. The M−M distances observed in the AAP−bestatin complex and native AAP

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