Catalytic mechanism of inulinase from Arthrobacter sp S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K-m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K-m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme. (C) 2008 Elsevier Inc. All rights reserved.

The direct determination of rare earth elements in basaltic and related rocks using ICP-MS: Testing the efficiency of microwave oven sample decomposition procedures

Tests are described showing the results obtained for the determination of REE and the trace elements Rb, Y, Zr, Nb, Cs, Ba, Hf, Ta, Pb, Th and U with ICP-MS methodology for nine basaltic reference materials, and thirteen basalts and amphibolites from the mafic-ultramafic Niquelandia Complex, central Brazil. Sample decomposition for the reference materials was performed by microwave oven digestion (HF and HNO(3), 100 mg of sample), and that for the Niquelandia samples also by Parr bomb treatment (5 days at 200 degrees C, 40 mg of sample). Results for the reference materials were similar to published values, thus showing that the microwave technique can be used with confidence for basaltic rocks. No fluoride precipitates were observed in the microwave-digested solutions. Total recovery of elements, including Zr and Hf, was obtained for the Niquelandia samples, with the exception of an amphibolite. For this latter sample, the Parr method achieved a total digestion, but not so the microwave decomposition; losses, however, were observed only for Zr and Hf, indicating difficulty in dissolving Zr-bearing minerals by microwave acid attack.

Towards a maximal extension of Pelczynski`s decomposition method in Banach spaces

l Suppose that X, Y. A and B are Banach spaces such that X is isomorphic to Y E) A and Y is isomorphic to X circle plus B. Are X and Y necessarily isomorphic? In this generality. the answer is no, as proved by W.T. Cowers in 1996. In the present paper, we provide a very simple necessary and sufficient condition on the 10-tuples (k, l, m, n. p, q, r, s, u, v) in N with p+q+u >= 3, r+s+v >= 3, uv >= 1, (p,q)$(0,0), (r,s)not equal(0,0) and u=1 or v=1 or (p. q) = (1, 0) or (r, s) = (0, 1), which guarantees that X is isomorphic to Y whenever these Banach spaces satisfy X(u) similar to X(p)circle plus Y(q), Y(u) similar to X(r)circle plus Y(s), and A(k) circle plus B(l) similar to A(m) circle plus B(n). Namely, delta = +/- 1 or lozenge not equal 0, gcd(lozenge, delta (p + q - u)) divides p + q - u and gcd(lozenge, delta(r + s - v)) divides r + s - v, where 3 = k - I - in + n is the characteristic number of the 4-tuple (k, l, m, n) and lozenge = (p - u)(s - v) - rq is the discriminant of the 6-tuple (p, q, r, s, U, v). We conjecture that this result is in some sense a maximal extension of the classical Pelczynski`s decomposition method in Banach spaces: the case (1, 0. 1, 0, 2. 0, 0, 2. 1. 1). (C) 2009 Elsevier Inc. All rights reserved.

Generalizations of Pelczynski`s decomposition method for Banach spaces containing a complemented copy of their squares

Suppose that X and Y are Banach spaces isomorphic to complemented subspaces of each other. In 1996, W. T. Gowers solved the Schroeder- Bernstein Problem for Banach spaces by showing that X is not necessarily isomorphic to Y. However, if X-2 is complemented in X with supplement A and Y-2 is complemented in Y with supplement B, that is, { X similar to X-2 circle plus A Y similar to Y-2 circle plus B, then the classical Pelczynski`s decomposition method for Banach spaces shows that X is isomorphic to Y whenever we can assume that A = B = {0}. But unfortunately, this is not always possible. In this paper, we show that it is possible to find all finite relations of isomorphism between A and B which guarantee that X is isomorphic to Y. In order to do this, we say that a quadruple (p, q, r, s) in N is a P-Quadruple for Banach spaces if X is isomorphic to Y whenever the supplements A and B satisfy A(p) circle plus B-q similar to A(r) circle plus B-s . Then we prove that (p, q, r, s) is a P-Quadruple for Banach spaces if and only if p - r = s - q = +/- 1.

The Catalytic Acid in the Dephosphorylation of the Cdk2-pTpY/CycA Protein Complex by Cdc25B Phosphatase

The development of anticancer therapeutics that target Cdc25 phosphatases is now an active area of research. A complete understanding of the Cdc25 catalytic mechanism would certainly allow a more rational inhibitor design. However, the identity of the catalytic acid used by Cdc25 has been debated and not established unambiguously. Results of molecular dynamics simulations with a calibrated hybrid potential for the first reaction step catalyzed by Cdc25B in complex with its natural substrate, the Cdk2-pTpY/CycA protein complex, are presented here. The calculated reaction free-energy profiles are in very good agreement with experimental measurements and are used to discern between different proposals for the general acid. In addition, the simulations give useful insight on interactions that can be explored for the design of inhibitors specific to Cdc25.

CeO(2)-catalyzed ozonation of phenol

Three different cerium citrate-based precursors were used for synthesizing CeO(2) through thermal treatment. Three morphological types of CeO(2) were obtained. Characterization of these oxides was carried out by XRD patterns, SEM microscopy, N(2) adsorption isotherms, Raman spectroscopy, zeta potential, and UV/Vis luminescence. Ozonation of phenol catalyzed by CeO(2) was studied as a representative reaction of environmental interest. The differences on the catalytic activity showed by these three oxides could be correlated to amounts of Ce(3+) on CeO(2) surface and, consequently, to the demand for oxygen needed to burn each precursor.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.

Experimental Evidence of the Occurrence of Intramolecular Electron Transfer in Catalyzed 1,2-Dioxetane Decomposition

The activation parameters for the thermal decomposition of 13 acridinium-substituted 1,2-dioxetanes, bearing an aromatic moiety, were determined and their chemiluminescence emission quantum yields estimated, utilizing in situ photosensitized 1,2-dioxetane generation and observation of its thermal decomposition kinetics, without isolation of these highly unstable cyclic peroxides. Decomposition rate constants show linear free-energy correlation for electron-withdrawing substituents, with a Hammett reaction constant of rho = 1.3 +/- 0.1, indicating the occurrence of an intramolecular electron transfer from the acridinium moiety to the 1,2-dioxetane ring, as postulated by the intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Emission quantum yield behavior can also be rationalized on the basis of the intramolecular CIEEL mechanism, additionally evidencing its occurrence in this transformation. Both relations constitute the first experimental evidence for the occurrence of the postulated intramolecular electron transfer in the catalyzed and induced decomposition of properly substituted 1,2-dioxetanes.

Thermal analysis of prednicarbate and characterization of thermal decomposition product

In the present work, the thermal behavior of prednicarbate was studied using DSC and TG/DTG. The solid product remaining at the first decomposition step of the drug was isolated by TG, in air and N(2) atmospheres and was characterized using LC-MS/MS, NMR, and IR spectroscopy. It was found that the product at the first thermal decomposition step of prednicarbate corresponds to the elimination of the carbonate group bonding to C(17), and a consequent formation of double bond between C(17) and C(16). Structure elucidation of this degradation product by spectral data has been discussed in detail.

Direct Kinetic Observation of the Chemiexcitation Step in Peroxyoxalate Chemiluminescence

A high-energy intermediate in the peroxyoxalate reaction can be accumulated at room temperature under specific reaction conditions and in the absence of any reducing agent in up to micromolar concentrations. Bimolecular interaction of this intermediate, accumulated in the reaction of oxalyl chloride with hydrogen peroxide, with an activator (highly fluorescent aromatic hydrocarbons with low oxidation potential) added in delay shows unequivocally that this intermediate is responsible for chemiexcitation of the activator. Activation parameters for the unimolccular decomposition of this intermediate (Delta H(double dagger) = 11.2 kcal mol(-1); Delta S(double dagger) = -23.2 cal mol(-1) K(-1)) and for its bimolecular reaction with 9,10-diphenylanthracene (Delta H(double dagger) = 4.2 kcal mol(-1); Delta S(double dagger) = -26.9 cal mol(-1) K(-1)) show that this intermediate is much less stable than typical 1,2-dioxetanes and 1,2-dioxetanones and demonstrate its highly favored interaction with the activator. Therefore, it can be inferred that structural characterization of the high-energy intermediate in the presence of an activator must be highly improbable. The observed linear free-energy correlation between the catalytic rate constants and the oxidation potentials of several activators definitely confirms the occurrence of the chemically initiated electron-exchange luminescence (CIEEL) mechanism in the chemiexcitation step of the peroxyoxalate system.

THERMAL BEHAVIOR STUDY AND DECOMPOSITION KINETICS OF SALBUTAMOL UNDER ISOTHERMAL AND NON-ISOTHERMAL CONDITIONS

The thermal decomposition of salbutamol (beta(2) - selective adrenoreceptor) was studied using differential scanning calorimetry (DSC) and thermogravimetry/derivative thermogravimetry (TG/DTG). It was observed that the commercial sample showed a different thermal profile than the standard sample caused by the presence of excipients. These compounds increase the thermal stability of the drug. Moreover, higher activation energy was calculated for the pharmaceutical sample, which was estimated by isothermal and non-isothermal methods for the first stage of the thermal decomposition process. For isothermal experiments the average values were E(act) = 130 kJ mol(-1) (for standard sample) and E(act) = 252 kJ mol(-1) (for pharmaceutical sample) in a dynamic nitrogen atmosphere (50 mL min(-1)). For non-isothermal method, activation energy was obtained from the plot of log heating rates vs. 1/T in dynamic air atmosphere (50 mL min(-1)). The calculated values were E(act) = 134 kJ mol(-1) (for standard sample) and E(act) (=) 139 kJ mol(-1) (for pharmaceutical sample).

Catalytic oxidation of hydrocarbons by trinuclear mu-oxo-bridged ruthenium-acetate clusters: Radical versus non-radical mechanisms

The [Ru(3)O(H(3)CCO(2))(6)(py)(2)(L)]PF(6) clusters, where L=methanol or dimethyl sulfoxide, can be activated by peroxide or oxygen donor species, such as tert-butyl hydroperoxide (TBHP) or iodosylbenzene (PhIO), respectively, generating reactive intermediates of the type [Ru(3)(IV,IV,III)=0](+). In this way, they catalyse the oxidation of cyclohexane or cyclohexene by TBHP and PhIO, via oxygen atom transfer, rather than by the alternative oxygen radical mechanism characteristic of this type of complexes. In addition to their ability to perform efficient olefin epoxydation catalysis, these clusters also promote the cleavage of the C-H bond in hydrocarbons, resembling the oxidation catalysis by metal porphyrins. (C) 2008 Elsevier Inc. All rights reserved.

Synthesis, characterization and adsorption properties of porous mixed valent diruthenium(II,III)-terephthalate and diruthenium(II,III)-adipate polymers

Two porous mixed valent diruthenium(II,III)-dicarboxylate compounds have been prepared and characterized by spectroscopic methods, X-ray diffraction and thermogravimetry. Crystalline solids of [Ru(2)(tere)(2)Cl] center dot 3.5H(2)O (tere=terephthalate) and [Ru(2)(adip)(2)Cl] center dot 1.5H(2)O (adip=adipate) consist of extended chains in which polymeric layers of multiply metal-metal bonded [Ru(2)](5+) cores are bridged by dicarboxylate ligands in paddlewheel type geometries. Units of [Ru(2)(dicarboxylate)(2)](n)(+) are linked by axial bridging chloride ions generating three-dimensional networks. The polymers loose non-bonded water molecules at low temperatures but do not undergo thermal decomposition below 280-300 degrees C. Both of compounds exhibit high BET surface areas, [Ru(2)(tere)(2)Cl]: 235 m(2) g(-1) and [Ru(2)(adip)(2)Cl]: 281 m(2) g(-1), and occlude similar numbers of mol of N(2) per mol of metal. The terephthalate ligand generated an organized structure with supermicropores (total pore size of 0.24 cm(3) g(-1)) while the adipate ligand led to a mesoporous structure (total pore sizes of 0.47 cm(3) g(-1)) for the corresponding diruthenium(II,III)-dicarboxylate polymers. (c) 2008 Elsevier B.V. All rights reserved.

SYNTHESIS, CHARACTERIZATION AND THERMAL ANALYSIS OF 1:1 AND 2:3 LANTHANIDE(III) CITRATES

The synthesis and characterization of lanthanide(III) citrates with stoichiometries 1:1 and 2:3; [LnL center dot xH(2)O] and [Ln(2)(LH)(3)center dot 2H(2)O], Ln=La, Ce, Pr, Nd, Sm and Eu are reported. L stands for (C6O7H5)(3-) and LH for (C6O7H6)(2-). Infrared absorption spectra of both series evidence coordination of carboxylate groups through symmetric bridges or chelation. X-ray powder patterns show the amorphous character of [LnL center dot xH(2)O]. The compounds [Ln(2)LH(3)center dot 2H(2)O] are crystalline and isomorphous. Emission spectra of Eu compounds suggest C-2v symmetry for the coordination polyhedron of [LnL center dot xH(2)O] and C-4v for [Ln(2)(LH)(3)center dot 2H(2)O]. Thermal analyses (TG-DTG-DTA) were carried out for both series. The thermal analysis patterns of the two series are quite different and both fit in a 4-step model of thermal decomposition, with lanthanide oxides as final products.

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB=5,5`-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the AFTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability. (C) 2011 Elsevier Inc. All rights reserved.