Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family

Chevrier, Bernard; Schalk, C.; d'Orchymont, Hugues; Rondeau, J.-M.; Moras, Dino; Tarnus, Céline

doi:10.1016/s0969-2126(00)00030-7

Cited by 263 publications

(332 citation statements)

References 35 publications

Supporting

Mentioning

319

Contrasting

Order By: Relevance

“…On the other hand, alignments of the human ASPA sequence against carboxypeptidase A, carboxypeptidase G, and aminopeptidase have recently been reported (Makarova & Grishin 1999). Two zinc ions were reported to bind to aminopeptidase (Berman et al 2000,Chevrier et al 1994) and carboxypeptidase G2 (Berman et al 2000,Rowsell et al 1997. In these peptidases, the ligands were shared between the two zinc ions.…”

Section: Homology-based Modeling Of Aspamentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

View full text Add to dashboard Cite

Mutations that result in undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-L-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

show abstract

Section: Homology-based Modeling Of Aspamentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

View full text Add to dashboard Cite

show abstract

“…8,9 On the basis of sequence alignments with other aminopeptidases 10 and several DapE gene sequences, all of the residues that function as ligands in the dinuclear active site of those enzymes are strictly conserved in DapE from Haemophilus influenzae. Studies on the E134A-and E134D-altered DapE revealed that E134 acts as the general acid/base in the hydrolysis of the substrate and is absolutely required for catalytic activity.…”

mentioning

confidence: 99%

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

Gillner

Armoush

Holz

et al. 2009

Bioorganic & Medicinal Chemistry Letters

View full text Add to dashboard Cite

“…that contains two g atoms of Zn(II) per mol of polypeptide, is thermostable for several hours at 70°C, can be obtained in large quantities (Ͼ100 mg), and can be genetically manipulated (2,17). 2 AAP has been crystallographically characterized and possesses a ( -aqua)( -carboxylato)dizinc(II) core with a terminal carboxylate and histidine residue coordinated to each metal ion (19). Both zinc ions reside in a distorted tetrahedral coordination geometry with a Zn-Zn distance of 3.5 Å.…”

mentioning

confidence: 99%

“…This carboxylate residue, usually a glutamate, often forms a hydrogen bond to a water molecule that is also bound to an active site metal ion. The x-ray crystal structure of AAP reveals that an oxygen atom of Glu-151 forms a hydrogen bond with a water molecule that bridges the two Zn(II) ions (19). Glu-151 has been proposed to act as the proton shuttle during catalytic turnover, but no direct evidence has been reported to substantiate this hypothesis (22).…”

mentioning

confidence: 99%

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Bzymek

Holz

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K m ) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k cat and K m revealed a change in the pK a of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK a values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.

show abstract

Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family

Cited by 263 publications

References 35 publications

Mutational analysis of aspartoacylase: Implications for Canavan Disease

Mutational analysis of aspartoacylase: Implications for Canavan Disease

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Contact Info

Product

Resources

About