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Article
Inhibition of the Aminopeptidase from Aeromonas proteolytica by l-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis
Biochemistry
  • Carin Stamper, Brandeis University
  • Brian Bennett, Marquette University
  • Tanya Edwards, Utah State University
  • Richard C. Holz, Marquette University
  • Dagmar Ringe, Brandeis University
  • Gregory A. Petsko, Brandeis University
Document Type
Article
Language
eng
Format of Original
12 p.
Publication Date
6-1-2001
Publisher
American Chemical Society
Original Item ID
doi: 10.1021/bi0100891
Disciplines
Abstract
The nature of the interaction of the transition-state analogue inhibitor l-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a Ki of 6.6 μM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 Å resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an η-1,2-μ-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.
Comments

Biochemistry, Vol. 40, No. 24 (June 2001): 7035-7046. 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 Stamper, Brian Bennett, Tanya Edwards, Richard C. Holz, et al.. "Inhibition of the Aminopeptidase from Aeromonas proteolytica by l-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis" Biochemistry (2001) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/37/