Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.
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.
Stamper
C
C
Program in Biophysics and Structural Biology, Department of Biochemistry and Rosenstiel Basic Medical Research Center, Brandeis University, 415 South Street, Waltham, Massachusetts 02254, USA.
Bennett
B
B
Edwards
T
T
Holz
R C
RC
Ringe
D
D
Petsko
G
G
D000333:Aeromonas / Q000201:enzymology*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D063065:Organophosphonates / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D063065:Organophosphonates; D010455:Peptides; C094802:leucine phosphonic acid; D000626:Aminopeptidases; D007930:Leucine