Spectroscopic and thermodynamic characterization of the E151D and E151A altered leucine aminopeptidases from Aeromonas proteolytica

Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 angstroms in carboxypeptidase G2 and 3.30 and 3.63 angstroms in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV-vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M(-1) cm(-1) vs. 56 and 109 M(-1) cm(-1) for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g(perpendicular) = 2.35, g(parallel) = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) g(x,y) = 2.509, g(z) = 2.19, E/D = 0.19, A = 0.0069 cm(-1) and (ii) g(x,y) = 2.565, g(z) = 2.19, E/D = 0.20, A = 0.0082 cm(-1) indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with K(d) values elevated by factors of approximately 850 and approximately 24,000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.     

Structure and thermodynamic aspects of macrobicyclic polyether-metal ion interactions

The crystals structure of a K⁺-diptychand-15C5-18C6 iodide complex has been determined from X-ray data. The complexed bicyclic molecule crystallizes in the triclinic space group witha = 9.995(4) Å,b = 10.097(4) Å,c = 13.725(6) Å, = 90.12(3)°, = 93.62(4)°, = 97.56(3)°. The structure was solved using heavy atom methods and refined toR = 0.032 for 3262 independent reflections. In the crystal structure, the K⁺ lies between the two crown ether rings, and is coordinated by the nine donor atoms of the ligand molecule. The complexation properties of the ligand with K⁺ and Na⁺ were studied by titration calorimetry in 90% (v/v) MeOH/H₂O solution. The studies indicate the formation of 1 : 1 ligand : metal ion complexes in both solid state and solution.This paper is dedicated to the memory of the late Dr C. J. Pedersen.     

Reaction of Nitroxides with Sulfur Containing Compounds II [1]. Preparation of Nitroxides Bearing an Isothiocyanate Substituent in View of the Nitroxyl Group Reduction with Thiophosgene

 The reaction of thiophosgene with 2,2,6,6-tetramethylpiperidine-1-oxyl (used as a model nitroxyl radical) was examined. 2,2,6,6-Tetramethylpiperidine and 2,2,6,6-tetramethyl-1-hydroxypiperidine were identified as products. The reaction is not competitive with the reaction of thiophosgene with an amino group. Thus, three nitroxides with an isothiocyanate group were synthesized from thiophosgene and the nitroxides containing the amino substituent.     

A concerted mechanism for the transfer of the thiophosphinoyl group from aryl dimethylphosphinothioate esters to oxyanionic nucleophiles in aqueous solution

Earlier work on the hydrolysis of aryl phosphinothioate esters has led to contradictory mechanistic conclusions. To resolve this mechanistic ambiguity, we have measured linear free energy relationships (beta(nuc) and beta(lg)) and kinetic isotope effects for the reactions of oxyanions with aryl dimethylphosphinothioates. For the attack of nucleophiles on 4-nitrophenyl dimethylphosphinothioate, beta(nuc) = 0.47 +/- 0.05 for phenoxide nucleophiles (pK(a) < 11) and beta(nuc) = 0.08 +/- 0.01 for hydroxide and alkoxide nucleophiles (pK(a) >or= 11). Linearity of the plot in the range that straddles the pK(a) of the leaving group (4-nitrophenoxide, pK(a) 7.14) is indicative of a concerted mechanism. The much lower value of beta(nuc) for the more basic nucleophiles reveals the importance of a desolvation step prior to rate-limiting nucleophilic attack. The reactions of a series of substituted aryl dimethylphosphinothioate esters give the same value of beta(lg) with the nucleophiles HO(-) (beta= -0.54 +/- 0.03) and PhO(-) (beta = -0.52 +/- 0.09). A significantly better Hammett correlation is obtained with sigma(-) than with sigma or sigma degrees , as expected for a transition state involving rate-limiting cleavage of the P-OAr bond. The (18)O KIE at the position of bond fission ((18)k = 1.0124 +/- 0.0008) indicates the P-O bond is approximately 40% broken, and the (15)N KIE in the leaving group ((15)k = 1.0009 +/- 0.0003) reveals the nucleofuge carries about a third of a negative charge in the transition state. Thus, both the LFER and KIE data are consistent with a concerted reaction and disfavor a stepwise mechanism.     

Photooxidation of Methionine Derivatives by the 4-Carboxybenzophenone Triplet State in Aqueous Solution. Intracomplex Proton Transfer Involving the Amino Group

Abstract— Oxidation of the triplet state of 4-carboxybenzophenone (CB) by a series of five substituted methionines and three methionine-containing dipeptides was monitored under laser flash photolysis conditions in aqueous solution. Spectral resolution techniques were employed to follow the concentration profiles of the intermediates formed from the quenching events. From these concentration profiles, quantum yields for the intermediates were determined. Branching ratios were evaluated for the decay of the charge-transfer complex by the competing processes of back electron transfer, proton transfer and escape of radical ions. The relative prominence of these processes was discussed in terms of the proton-transfer tendencies of the nominal sulfur-radical-cationic species. A systematic decrease was observed in the quantum yields for the escape of radical ions along with a correlated increase in the proton-transfer yields. The enhanced propensity of the sulfur radical cations to depro-tonate is due to deprotonation at the carbons adjacent to the sulfur-cationic site and at the unsubstituted amino groups when present. This scheme was supported by an observed decrease in the yields of dimeric sulfur radical cations with an increase in the electron-withdrawing abilities of the substituents, making the radical-cationic species stronger acids. The involvement of protons on the amino groups was implicated by the correlation of the quantum yields of ketyl radical formation in the photochemistry experiments with the rate constants for the reaction of the CB radical anion with the sulfur-containing substrates in pulse radiolysis experiments.     

Separable quantizations of Stäckel systems

In this article we prove that many Hamiltonian systems that cannot be separably quantized in the classical approach of Robertson and Eisenhart can be separably quantized if we extend the class of admissible quantizations through a suitable choice of Riemann space adapted to the Poisson geometry of the system. Actually, in this article we prove that for every quadratic in momenta Stäckel system (defined on 2n$2n$ dimensional Poisson manifold) for which Stäckel matrix consists of monomials in position coordinates there exist infinitely many quantizations–parametrized by n$n$ arbitrary functions–that turn this system into a quantum separable Stäckel system.     

Hydrolates from lavender (Lavandula angustifolia) – their chemical composition as well as aromatic,antimicrobial and antioxidant properties

It was shown that the method for obtaining hydrolates from lavender (Lavandula angustifolia) influences the content of active compounds and the aromatic, antimicrobial and antioxidant properties of the hydrolates. The content of volatile organic compounds ranged from 9.12 to 97.23 mg/100 mL of hydrolate. Lavender hydrolate variants showed low antimicrobial activity (from 0% to 0.05%). The radical scavenging activity of DPPH was from 3.6 ± 0.5% to 3.8 ± 0.6% and oxygen radical absorbance capacity (ORAC_FL) results were from 0 to 266 μM Trolox equivalent, depending on the hydrolate variant.     

On an equation characterizing multi-Jensen-quadratic mappings and its Hyers–Ulam stability via a fixed point method

In this paper, we unify the system of functional equations defining a multi-Jensen-quadratic mapping to obtain a single equation. We also prove, using the fixed point method, the generalized Hyers–Ulam stability of this equation both in Banach spaces and in complete non-Archimedean normed spaces.     

On Hyers–Ulam stability of two functional equations in non-Archimedean spaces

In this paper we prove, using the fixed point method, the generalized Hyers–Ulam stability of two functional equations in complete non-Archimedean normed spaces. One of these equations characterizes multi-Cauchy–Jensen mappings, and the other gives a characterization of multi-additive-quadratic mappings.