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

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [ 3 H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [ 3 H]inositol phosphates (mainly inositol monophos-phate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca 2+ and was maximal at 0.2 m M Ca 2+ . Increasing extracellular Ca 2+ from 0.22 to 2.2 m M increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K + also stimulated Ca 2+ -dependent [ 3 H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca 2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K + to a greater extent than that of muscarine. Ca 2+ (0.3–10 Μ M ) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [ 3 H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K + probably increased the accumulation of inositol phosphates through Ca 2+ influx and a rise in cytosolic Ca 2+ . Because Ba 2+ caused catechol-amine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and wyo -inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca 2+ from intracellular stores.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65738/1/j.1471-4159.1987.tb01037.x.pd

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-Å resolution at neutral pH and at 1.24-Å 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 Å 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 Å, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Å, respectively. A second-shell feature at ca. 3.34 Å 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

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

Fast 3D Perception for Collision Avoidance and SLAM in Domestic Environments

Electronics engineerin

Chapter Fast 3D Perception for Collision Avoidance and SLAM in Domestic Environments

Electronics engineerin