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David L. Bienvenue

Bio: David L. Bienvenue is an academic researcher from Utah State University. The author has contributed to research in topics: Active site & Binding site. The author has an hindex of 10, co-authored 11 publications receiving 300 citations. Previous affiliations of David L. Bienvenue include Medical College of Wisconsin & Loyola University Chicago.

Papers
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Journal ArticleDOI
TL;DR: To fully understand the metal roles in the reaction pathway of AAP, the 1.20 A resolution crystal structure of native AAP is solved and insight is led to into the protonation states of some of the active site amino acid side chains.

54 citations

Journal ArticleDOI
TL;DR: The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalentMetal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.
Abstract: The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to approximately 20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) approximately equal Cu(II) approximately equal Mg(II). DapE was shown to only hydrolyze L,L-N-succinyl-diaminopimelic acid (L,L-SDAP) and was inactive toward D,L-, L,D-, and D,D-SDAP. DapE was also inactive toward several acetylated amino acids as well as D,L-succinyl aminopimelate, which differs from the natural substrate, L,L-SDAP, by the absence of the amine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 x 10(3) times (K(d1) = 0.14 microM vs K(d2) = 300 microM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degrees C and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 degrees C being DeltaG(++) = 64 kJ/mol, DeltaH(++) = 28.5 kJ/mol, and DeltaS(++) = -119 J mol(-1) K(-1). Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin-spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalent metal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.

42 citations

Journal ArticleDOI
TL;DR: The crystal structures of AAP are reported at 0.95-Å resolution at neutral pH and at low pH to allow the precise modeling of atomic positions, the identification of theMetal bridging oxygen species, and insight into the physical properties of the metal ions.
Abstract: The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-A resolution at neutral pH and at 1.24-A resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.

34 citations

Journal ArticleDOI
TL;DR: Spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design.
Abstract: Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two Co(II) ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom bridges between the two divalent metal ions. The EPR data obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex was solved to 2.0 A resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active site. The amino acid residues ligated to the dizinc(II) cluster in AAP are identical to those in the native structure with only minor perturbations in bond length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in the active site, displacing the bridging water molecule observed in the native [ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1 and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In addition, the NH(2) group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A combination of the spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design.

33 citations

Journal ArticleDOI
TL;DR: In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue and E134 is intrinsically involved in the hydrolysis reaction catalyzed by DAPE and likely plays the role of a general acid and base.
Abstract: Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (Km) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine (0%). Examination of the pH dependence of the kinetic constants kcat and Km for E134D-DapE revealed ionizations at pH 6.4, 7.4, and approximately 9.7. Isothermal titration calorimetry experiments revealed a significant weakening in metal Kd values of E134D-DapE. D134 and A134 perturb the second divalent metal binding site significantly more than the first, but both altered enzymes can still bind two divalent metal ions. Structural perturbations of the dinuclear active site of DapE were also examined for two E134-substituted forms, namely E134D-DapE and E134A-DapE, by UV–vis and electron paramagnetic resonance (EPR) spectroscopy. UV–vis spectroscopy of Co(II)-substituted E134D-DapE and E134A-DapE did not reveal any significant changes in the electronic absorption spectra, suggesting that both Co(II) ions in E134D-DapE and E134A-DapE reside in distorted trigonal bipyramidal coordination geometries. EPR spectra of [Co_(E134D-DapE)] and [Co_(E1341A-DapE] are similar to those observed for [CoCo(DapE)] and somewhat similar to the spectrum of [Co(H2O)6]2+ which typically exhibit E/D values of approximately 0.1. Computer simulation returned an axial g-tensor with g(x,y)=2.24 and E/D=0.07; gz was only poorly determined, but was estimated as 2.5–2.6. Upon the addition of a second Co(II) ion to [Co_(E134D-DapE)] and [Co_(E134A-DapE)], a broad axial signal was observed; however, no signals were observed with B0||B1 (“parallel mode”). On the basis of these data, E134 is intrinsically involved in the hydrolysis reaction catalyzed by DapE and likely plays the role of a general acid and base.

28 citations


Cited by
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Journal ArticleDOI
TL;DR: The contributions of many laboratories as they have sought to understand the molecular basis for substrate specificity and catalysis are described.
Abstract: Metalloaminopeptidases remove the N-terminal amino acid from proteins or small oligopeptides. This modification or truncation process can occur either co-translationally or posttranslationally after the action of an endoproteinase. The action or inaction of these exopeptidases is instrumental for many normal and pathophysiological processes. The metalloaminopeptidases utilize conserved amino acid residues to generate a scaffold capable of binding one or two metal ions. The metal centers can be described as [M1M2(enzyme)], where M1 and M2 indicate the type of metal ion in site 1 and site 2 for a particular enzyme. Spectroscopic and kinetic analyses indicate that for some of the enzymes two metal ions are required for full activity. In other instances, only a single metal ion is essential for catalysis while the second metal ion modulates activity either positively or negatively. These mononuclear and cocatalytic,1 dinuclear metal centers mediate catalysis by providing sites for substrate binding, by activating the nucleophile of the reaction, and by stabilizing the transition state. Structural analyses of representatives of the different metalloaminopeptidase classes indicate that the site 1 metal ion is closest to the mouth of the active site while the site 2 ion is relatively buried and adjacent to the binding pocket for the P1 side chain (nomenclature of Schechter and Berger2). The enzymes can be categorized based on their substrate specificity or preferences.3 Cleavage of the scissile peptide bond is dependent upon the chemical structure and composition of the amino acid residues that flank the cleavage site (P1*P1′). The broad-range aminopeptidases are typified by the bovine lens leucine aminopeptidase (bLeuAP) and the aminopeptidases from Aeromonas proteolytica (ApAP) and Streptomyces griseus (SgAP). These enzymes are capable of cleaving hydrophobic amino acids from the N-terminus, usually without strict requirements for the penultimate (P1′) residue. In contrast, the “pitabread” enzymes methionine aminopeptidase (MetAP), aminopeptidase P (APP), and prolidase process substrates restricted to particular N-terminal and penultimate residues. This review describes the contributions of many laboratories as they have sought to understand the molecular basis for substrate specificity and catalysis. An effort has been made to give sufficient historical background to appreciate the current state of knowledge. The structure-function relationships for each § Present address: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, WinstonSalem, NC 27157 4581 Chem. Rev. 2002, 102, 4581−4607

315 citations

Journal ArticleDOI
TL;DR: A Law of Matching Water Affinities is approximateed and used to examine the issues of how ions are selected to be compatible with the high solubility requirements of cytosolic components and the "chelate effect" is used by macromolecules to bind ions at specific sites even when there is a mismatch in water affinity.

314 citations

Journal ArticleDOI
TL;DR: The 3.5-Å crystal structure of the PSMA ectodomain is presented, which reveals a homodimer with structural similarity to transferrin receptor, a receptor for iron-loaded transferrin that lacks protease activity.
Abstract: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer cells and nonprostatic solid tumor neovasculature and is a target for anticancer imaging and therapeutic agents. PSMA acts as a glutamate carboxypeptidase (GCPII) on small molecule substrates, including folate, the anticancer drug methotrexate, and the neuropeptide N-acetyl-l-aspartyl-l-glutamate. Here we present the 3.5-A crystal structure of the PSMA ectodomain, which reveals a homodimer with structural similarity to transferrin receptor, a receptor for iron-loaded transferrin that lacks protease activity. Unlike transferrin receptor, the protease domain of PSMA contains a binuclear zinc site, catalytic residues, and a proposed substrate-binding arginine patch. Elucidation of the PSMA structure combined with docking studies and a proposed catalytic mechanism provides insight into the recognition of inhibitors and the natural substrate N-acetyl-l-aspartyl-l-glutamate. The PSMA structure will facilitate development of chemotherapeutics, cancer-imaging agents, and agents for treatment of neurological disorders.

249 citations

Journal ArticleDOI
TL;DR: An overview of the literature for 2005 is attempted to highlight works of interest and novelty and draw attention to those works which it is felt have provided a route to better analysis and increased the ability to understand the meaning of thermodynamic change on binding.
Abstract: Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naive, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding.

191 citations