33 research outputs found
Inhibition of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e by l-leucinethiol: Kinetic and Spectroscopic Characterization of a Slow, Tight-binding Inhibitor–enzyme Complex
The peptide inhibitor l-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency (KI*) of LeuSH was 7 nM while the corresponding alcohol l-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (KI=17 μM). These data suggest that the free thiol is likely involved in the formation of the E·I and E·I* complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] in the presence of both inhibitors. In the presence of LeuSH, all three Co(II)-substituted AAP enzymes exhibited an absorption band centered at 295 nm, characteristic of a S→Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded in the 450 to 700 nm region all showed changes characteristic of LeuSH and LeuOH interacting with both metal ions. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that, in a given enzyme molecule, LeuSH interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified μ-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon binding LeuSH. EPR spectra of [CoCo(AAP)]-LeuSH, [ZnCo(AAP)]-LeuSH, and [Co_(AAP)]-LeuSH were also recorded at lower temperature (3.5–4.0 K) and high microwave power (50–553 mW). These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at geff∼6.5 that is characteristic of thiolate-Co(II) interactions. Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both metal ions in the dinuclear active site of AAP. Since the spin–spin interaction seen in resting [CoCo(AAP)] is abolished upon the addition of LeuSH, it is unlikely that a μ-S(R) bridge is established
Hydrolysis of Thionopeptides by the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e:  Insight into Substrate Binding
A series of l-leucine aniline analogues were synthesized that contained either a carbonyl or thiocarbonyl as a part of the amide bond. Additionally, the para-position on the phenyl ring of several substrates was altered with various electron-withdrawing or donating groups. The kinetic constants Km and kcat were determined for the hydrolysis of each of these compounds in the presence of the aminopeptidase from Aeromonas proteolytica (AAP) containing either Zn(II) or Cd(II). The dizinc(II) form of AAP ([ZnZn(AAP)]) was able to cleave both carbonyl and thiocarbonyl containing peptide substrates with similar efficiency. However, the dicadmium(II) form of AAP ([CdCd(AAP)]) was unable to cleave any of the carbonyl-containing compounds tested but was able to cleave the thionopeptide substrates. This is consistent with the borderline hard/soft nature of Zn(II) vs Cd(II). The trends observed in the Km values suggest that the oxygen atom of the amide bond directly interacts with the dinuclear active site of AAP. Heterodimetallic forms of AAP that contained one atom of Zn(II) and one of Cd(II) (i.e., [CdZn(AAP)] and [ZnCd(AAP)]) were also prepared. The Km values for the thionopeptides substrates are the smallest when Cd(II) is in the first metal binding site, suggesting that substrate binds to the first metal binding site. 1-Phenyl-2-thiourea (PTU) and urea (PU) were also examined to determine the differences between thionopeptide and peptide binding to AAP. PTU and PU were found to be competitive inhibitors of AAP with inhibition constants of 0.24 and 4.6 mM, respectively. The electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] were recorded in the absence and presence of both PU and PTU. Spectral changes were observed for PTU binding to [CoCo(AAP)] and [CoZn(AAP)] but not for [ZnCo(AAP)], while no spectral changes were observed for any of the Co(II)-substituted forms of AAP upon the addition of PU. These data indicate that carbonyl binding occurs only at the first metal binding site. In light of the data presented herein, the substrate binding step in the proposed mechanism of AAP catalyzed peptide hydrolysis can be further refined
The High-Resolution Structures of the Neutral and the Low pH Crystals of Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e
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-Ã… resolution at neutral pH and at 1.24-Ã… 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
The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-succinyl-l,l-diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Contains Two Active-site Histidine Residues
The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Ã… of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical
The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Is a Dinuclear Metallohydrolase
The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 Ã…, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Ã…, respectively. A second-shell feature at ca. 3.34 Ã… appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented
Fighting Antibiotic Resistance: The \u3cem\u3edapE\u3c/em\u3e-encoded N-succinyl-L, L-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e in a Dinuclear Metallohydrolase
Substrate Specificity, Metal Binding Properties, and Spectroscopic Characterization of the DapE-Encoded N-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e
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 ∼20% of the Zn(II)-loaded enzyme. The order of the observed kcat values are Co(II) \u3e Zn(II) \u3e Cd(II) \u3e Mn(II) \u3eNi(II) ∼ Cu(II) ∼ 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 × 103 times (Kd1 = 0.14 μM vs Kd2 = 300 μM). In addition, an Arrhenius plot was constructed from kcat values measured between 16 and 35 °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 °C being ΔG⧧ = 64 kJ/mol, ΔH⧧ = 28.5 kJ/mol, and ΔS⧧ = −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
Slow-Binding Inhibition of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e by Peptide Thiols: Synthesis and Spectroscopic Characterization
Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a−c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (KI*) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a−b) are simple competitive inhibitors of much lower potencies (KI = 23 and 360 μM). These data suggest that the free thiols are involved in the formation of the E·I and E·I* complexes, presumably serving as a metal ligand. To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c. Both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S → Co(II) ligand−metal charge-transfer band. In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified μ-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding. EPR spectra of [CoCo(AAP)]-1c, [ZnCo(AAP)]-1c, and [CoZn(AAP)]-1c were also recorded at lower temperature (3.5−4.0 K) and high microwave power (50−553 mW). The observed signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at geff ≈ 6.8 that is characteristic of thiolate-Co(II) interactions. These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster. Compounds 1a−c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors
Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism
The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn–Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes
Spectroscopic and X-ray Crystallographic Characterization of Bestatin Bound to the Aminopeptidase from \u3cem\u3eAeromonas (Vibrio) proteolytica\u3c/em\u3e
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 CoII 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 Å resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear ZnII 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 ZnII 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 Å) with alkoxide oxygen atom distances of 2.1 and 1.9 Å from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 Å. In addition, the NH2 group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 Å. 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