Document Type




Format of Original

9 p.

Publication Date



American Chemical Society

Source Publication


Source ISSN


Original Item ID

doi: 10.1021/bi049126p


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.


Accepted version. Biochemistry, Vol. 43, No. 30 (August 2004): 9620-9628. DOI. © 2004 American Chemical Society Publications. Used with permission.

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.

bennett_6059acc.docx (213 kB)
ADA Accessible Version

Included in

Physics Commons