Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis. Biochemistry 2001 Jun 19;40(24):7035-46
Date
06/13/2001Pubmed ID
11401547DOI
10.1021/bi0100891Scopus ID
2-s2.0-0035912827 (requires institutional sign-in at Scopus site) 72 CitationsAbstract
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 K(i) of 6.6 microM. 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 A 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 eta-1,2-mu-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.
Author List
Stamper C, Bennett B, Edwards T, Holz RC, Ringe D, Petsko GAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
AeromonasAminopeptidases
Binding, Competitive
Catalysis
Crystallization
Crystallography, X-Ray
Electron Spin Resonance Spectroscopy
Enzyme Inhibitors
Hydrolysis
Kinetics
Leucine
Macromolecular Substances
Organophosphonates
Peptides
Protein Binding
Spectrophotometry, Ultraviolet
Thermodynamics