Inhibition of the Aminopeptidase from <i>Aeromonas proteolytica</i> by <scp>l</scp>-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis

Stamper, Carin C.; Bennett, Brian; Edwards, Tanya; Holz, Richard C.; Ringe, Dagmar; Petsko, Gregory A.

doi:10.1021/bi0100891

Cited by 79 publications

(132 citation statements)

References 56 publications

Supporting

Mentioning

122

Contrasting

Order By: Relevance

“…Deprotonation of the metal-bound water molecule to form a nucleophilic hydroxide moiety is particularly important at pH values lower than 7.0, the postulated pK a of the bridging water molecule, yet appears not to be the ratelimiting step based on higher stability of the -hydroxo bridge (18). Once the metal-bound hydroxide is formed, it can attack the activated carbonyl carbon of the peptide substrate forming a gem-diolate transition state complex that is stabilized by coordination of both oxygen atoms to the dizinc(II) center, consistent with the x-ray crystal structure of [ZnZn(AAP)] bound by L-leucine phosphonic acid (21). Glu-151 then provides an additional proton to the newly formed N-terminal nitrogen moiety.…”

Section: Temperature Dependence Of K Cat and K M For E151d-aap-supporting

confidence: 60%

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

Bzymek

Holz

2004

Journal of Biological Chemistry

Self Cite

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

Section: Temperature Dependence Of K Cat and K M For E151d-aap-supporting

confidence: 60%

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

Bzymek

Holz

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The metal ligands are identical to those present in AAP (37) and PhTET2 (19,20), with an identical type of coordination. Therefore, the catalytic mechanism of hydrolysis by AAP, PhTET2, and PhTET1 should be extremely similar, if not identical (19,20,40).…”

Section: Resultsmentioning

confidence: 99%

An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Schoehn

Vellieux

Durá

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.Cytosolic proteolysis is a key cellular process. In addition to its role in amino acid metabolism, it is responsible for protein quality control and the adjustment of the turnover of regulatory molecules (1, 2). Initial proteolysis is carried out by nonspecific endoproteases (1). Through oligomerization, these enzymes form barrel-like cellular subcompartments to protect cytoplasmic proteins from unwanted cleavage; the active sites are situated in proteolytic chambers, only accessible to unfolded polypeptides. In Eukarya and Archaea, the 20 S proteasome is the core of this proteolytic system (3, 4). The proteins to be degraded are unfolded and translocated into the proteolytic complex by energy-dependent regulatory particles (5). Polypeptide degradation results in multiple endoproteolytic cleavage, yielding short peptides (6 -12 residues in length) (6). The final breakdown of these fragments is probably achieved by a collection of ATP-independent exopeptidases, which have been proposed to be essential components of the protein degradation system (7,8).Structural studies on the Tricorn protease complex, an archaeal carboxypeptidase (peptidyl-di/tripeptidase), have shown that some exopeptidases can also form multimeric ring structures, with the active sites sequestered within an internal chamber (9). This feature of compartmentalization was also recognized in the three-dimensional structures of three ATP-independent intracellular aminopeptidases: Gal6, leucine aminopeptidase, and the D-aminopeptidase DppA (10 -12). In eukaryotic cells, the tripeptidyl-peptidase II also forms a giant, well organized toroidal oligomeric structure (13). All these exopeptidases could function as scavengers of the oligopeptides generated by the ATP-dependent endoproteases (14).TET is another type of a large hollow peptidase complex. It was characterized initially in the halophilic Archaea Haloarcula marismortui (15). The complex has a tetrahedral shape and is a broad substrate specificity ...

show abstract

“…Thiol and carboxylic acid groups are also essential units in captopril and enalaprilat, representative angiotensin-converting enzyme inhibitors that are used for the treatment of hypertension [3,18]. Phosphonate and boronate groups are mimics of the tetrahedral transition state of amide hydrolysis [19][20][21]. L-Leucine phosphonate (LPA) is also known to bind to Zn 2?…”

Section: Introductionmentioning

confidence: 99%

Potent inhibition of dinuclear zinc(II) peptidase, an aminopeptidase from Aeromonas proteolytica, by 8-quinolinol derivatives: inhibitor design based on Zn2+ fluorophores, kinetic, and X-ray crystallographic study

et al. 2012

View full text Add to dashboard Cite

The selective inhibition of an aminopeptidase from Aeromonas proteolytica (AAP), a dinuclear Zn(2+) hydrolase, by 8-quinolinol (8-hydroxyquinoline, 8-HQ) derivatives is reported. We previously reported on the preparation of 8-HQ-pendant cyclens as Zn(2+) fluorophores (cyclen is 1,4,7,10-tetraazacyclododecane), in which the nitrogen and phenolate of the 8-HQ units (as well as the four nitrogens of cyclen) bind to Zn(2+) in a bidentate manner to form very stable Zn(2+) complexes at neutral pH (K (d) = 8-50 fM at pH 7.4). On the basis of this finding, it was hypothesized that 8-HQ derivatives have the potential to function as specific inhibitors of Zn(2+) enzymes, especially dinuclear Zn(2+) hydrolases. Assays of 8-HQ derivatives as inhibitors were performed against commercially available dinuclear Zn(2+) enzymes such as AAP and alkaline phosphatase. 8-HQ and the 5-substituted 8-HQ derivatives were found to be competitive inhibitors of AAP with inhibition constants of 0.16-29 μM at pH 8.0. The nitrogen at the 1-position and the hydroxide at the 8-position of 8-HQ were found to be essential for the inhibition of AAP. Fluorescence titrations of these drugs with AAP and an X-ray crystal structure analysis of an AAP-8-HQ complex (1.3-Å resolution) confirmed that 8-HQ binds to AAP in the "Pyr-out" mode, in which the hydroxide anion of 8-HQ bridges two Zn(2+) ions (Zn1 and Zn2) in the active site of AAP and the nitrogen atom of 8-HQ coordinates to Zn1 (Protein Data Bank code 3VH9).

show abstract

Inhibition of the Aminopeptidase from Aeromonas proteolytica by l-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis

Cited by 79 publications

References 56 publications

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

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

An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Potent inhibition of dinuclear zinc(II) peptidase, an aminopeptidase from Aeromonas proteolytica, by 8-quinolinol derivatives: inhibitor design based on Zn2+ fluorophores, kinetic, and X-ray crystallographic study

Contact Info

Product

Resources

About