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Article
Spectroscopic and X-ray Crystallographic Characterization of Bestatin Bound to the Aminopeptidase from Aeromonas (Vibrio) proteolytica
Biochemistry
  • Carin C. Stamper, Brandeis University
  • David L. Bienvenue, Utah State University
  • Brian Bennett, Marquette University
  • Dagmar Ringe, Brandeis University
  • Gregory A. Petsko, Brandeis University
  • Richard C. Holz, Marquette University
Document Type
Article
Language
eng
Format of Original
9 p.
Publication Date
8-1-2004
Publisher
American Chemical Society
Original Item ID
doi: 10.1021/bi049126p
Disciplines
Abstract
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 CoII 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 ZnII 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 ZnII 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 Å) with alkoxide oxygen atom distances of 2.1 and 1.9 Å from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 Å. In addition, the NH2 group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 Å. 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.
Comments

Biochemistry, Vol. 43, No. 30 (August 2004): 9620-9628. DOI.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

Richard Holz was affiliated with Utah State University at the time of publication.

Citation Information
Carin C. Stamper, David L. Bienvenue, Brian Bennett, Dagmar Ringe, et al.. "Spectroscopic and X-ray Crystallographic Characterization of Bestatin Bound to the Aminopeptidase from Aeromonas (Vibrio) proteolytica" Biochemistry (2004) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/46/