20 resultados para Metalloenzyme


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The prime thrust of this dissertation is to advance the development of fuel cell dioxygen reduction cathodes that employ some variant of multicopper oxidase enzymes as the catalyst. The low earth-abundance of platinum metal and its correspondingly high market cost has prompted a general search amongst chemists and materials scientists for reasonable alternatives to this metal for facilitating catalytic dioxygen reduction chemistry. The multicopper oxidases (MCOs), which constitute a class of enzyme that naturally catalyze the reaction O2 + 4H+ + 4e- → 2H2O, provide a promising set of biochemical contenders for fuel cell cathode catalysts. In MCOs, a substrate reduces a copper atom at the type 1 site, where charge is then transferred to a trinuclear copper cluster consisting of a mononuclear type 2 or “normal copper” site and a binuclear type 3 copper site. Following the reduction of all four copper atoms in the enzyme, dioxygen is then reduced to water in two two-electron steps, upon binding to the trinuclear copper cluster. We identified an MCO, a laccase from the hyperthermophilic bacterium Thermus thermophilus strain HB27, as a promising candidate for cathodic fuel cell catalysis. This protein demonstrates resilience at high temperatures, exhibiting no denaturing transition at temperatures high as 95°C, conditions relevant to typical polymer electrolyte fuel cell operation.

In Chapter I of this thesis, we discuss initial efforts to physically characterize the enzyme when operating as a heterogeneous cathode catalyst. Following this, in Chapter II we then outline the development of a model capable of describing the observed electrochemical behavior of this enzyme when operating on porous carbon electrodes. Developing a rigorous mathematical framework with which to describe this system had the potential to improve our understanding of MCO electrokinetics, while also providing a level of predictive power that might guide any future efforts to fabricate MCO cathodes with optimized electrochemical performance. In Chapter III we detail efforts to reduce electrode overpotentials through site-directed mutagenesis of the inner and outer-sphere ligands of the Cu sites in laccase, using electrochemical methods and electronic spectroscopy to try and understand the resultant behavior of our mutant constructs. Finally, in Chapter IV, we examine future work concerning the fabrication of enhanced MCO cathodes, exploring the possibility of new cathode materials and advanced enzyme deposition techniques.

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The isomerization of glucose into fructose is a large-scale reaction for the production of high-fructose corn syrup, and is now being considered as an intermediate step in the possible route of biomass conversion into fuels and chemicals. Recently, it has been shown that a hydrophobic, large pore, silica molecular sieve having the zeolite beta structure and containing framework Sn4+ (Sn-Beta) is able to isomerize glucose into fructose in aqueous media. Here, I have investigated how this catalyst converts glucose to fructose and show that it is analogous to that achieved with metalloenzymes. Specifically, glucose partitions into the molecular sieve in the pyranose form, ring opens to the acyclic form in the presence of the Lewis acid center (framework Sn4+), isomerizes into the acyclic form of fructose and finally ring closes to yield the furanose product. Akin to the metalloenzyme, the isomerization step proceeds by intramolecular hydride transfer from C2 to C1. Extraframework tin oxides located within hydrophobic channels of the molecular sieve that exclude liquid water can also isomerize glucose to fructose in aqueous media, but do so through a base-catalyzed proton abstraction mechanism. Extraframework tin oxide particles located at the external surface of the molecular sieve crystals or on amorphous silica supports are not active in aqueous media but are able to perform the isomerization in methanol by a base-catalyzed proton abstraction mechanism. Post-synthetic exchange of Na+ with Sn-Beta alters the glucose reaction pathway from the 1,2 intramolecular hydrogen shift (isomerization) to produce fructose towards the 1,2 intramolecular carbon shift (epimerization) that forms mannose. Na+ remains exchanged onto silanol groups during reaction in methanol solvent, leading to a near complete shift in selectivity towards glucose epimerization to mannose. In contrast, decationation occurs during reaction in aqueous solutions and gradually increases the reaction selectivity to isomerization at the expense of epimerization. Decationation and concomitant changes in selectivity can be eliminated by addition of NaCl to the aqueous reaction solution. Thus, framework tin sites with a proximal silanol group are the active sites for the 1, 2 intramolecular hydride shift in the isomerization of glucose to fructose, while these sites with Na-exchanged silanol group are the active sites for the 1, 2 intramolecular carbon shift in epimerization of glucose to mannose.

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LL catalytic RNAs (ribozymes) require or are stimulated by divalent metal ions, but it has been difficult to separate the contribution of these metal ions to formation of the RNA tertiary structure1 from a more direct role in catalysis. The Tetrahymena ribozyme catalyses cleavage of exogenous RNA2,3 or DNA4,5 substrates with an absolute requirement for Mg2+ or Mn2+ (ref. 6). A DNA substrate, in which the bridging 3' oxygen atom at the cleavage site is replaced by sulphur, is cleaved by the ribozyme about 1,000 times more slowly than the corresponding unmodified DNA substrate when Mg2+ is present as the only divalent metal ion. But addition of Mn2+ or Zn2+ to the reaction relieves this negative effect, with the 3' S–P bond being cleaved nearly as fast as the 3' O–P bond. Considering that Mn2+ and Zn2+ coordinate sulphur more strongly than Mg2+ does7,8, these results indicate that the metal ion contributes directly to catalysis by coordination to the 3' oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group. We conclude that the Tetrahymena ribozyme is a metalloenzyme, with mechanistic similarities to several protein enzymes9–12.

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Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

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In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn(2+) applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni(2+) resin was directly proportional to the concentration of Mn(2+). Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni(2+), allowing the following to occur: (1) entrance of Mn(2+) and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni(2+) after the Mn(2+) activation step. The conformational alterations can be summarized as follows: the interaction with the Ni(2+) simulates thermal heating in the artificial activation by opening a channel for Mn(2+) to enter. (C) 2010 Elsevier Inc. All rights reserved.

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Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn2+ (sites I and III) and one for Mg2+ (site II). This enzyme is stimulated synergistically by Zn2+ and Mg2+ (Ciancaglini et al., 1992) and also by Mn2+ (Leone et al., 1995) and Co2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca2+. In the absence of Zn2+ and Mg2+, Ca2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 mu M MgCl2, increasing concentration of Ca2+ were inhibitory, suggesting the displacement of Mg2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn2+ concentrations Zip to 0.1 mu M were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K-0.5 = 8.5 nM. Above 0.1 mu M Zn2+ exerted a strong inhibitory effect and concentrations of Ca2+ up to I mM were not enough to counteract this inhibition, indicating that Ca2+ was easily displaced by Zn2+. At fixed concentrations of Ca2+, increasing concentrations of Mg2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K-0.5 values were significantly affected (from 4.4 mu M to 38.0 mu M). The synergistic effects observed for the activity of Ca2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca2+ on the activity of the enzyme is presented. (C) 1997 Elsevier B.V.

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Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.