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

NUNES, Genebaldo S.; ALEXIOU, Anamaria D. P.; TOMA, Henrique E.
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
Português
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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.

Isosorbide Polyesters from Enzymatic Catalysis

JUAIS, Danielle; NAVES, Alliny F.; LI, Chong; GROSS, Richard A.; CATALANI, Luiz H.
Fonte: AMER CHEMICAL SOC Publicador: AMER CHEMICAL SOC
Tipo: Artigo de Revista Científica
Português
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The synthesis of isosorbide aliphatic polyesters is demonstrated by the use of Novozym 435, a catalyst consisting of Candida antarctica lipase B immobilized on a macroporous support Several experimental procedures were tested and azeotropic distillation was most effective in removing low mass byproduct Furthermore, the use of diethyl ester derivatives of diacid comonomers gave isosorbide copolyesters with highest Isolated yield and molecular weights The length of the diacid aliphatic chain was less restrictive, but with a clear preference for longer aliphatic chains The molecular mass values of the obtained products were equivalent or higher than those obtained by nonenzymatic polymerizations, a clear illustration of the potential of enzymatic over conventional catalysis The ability of Novozym 435 to catalyze the synthesis of isosorbide polyester with weight-average molecular weights in excess of 40000 Da was unexpected given that isosorbide has two chemically distinct secondary hydroxyl groups This is the first example in which isosorbide polyesters were synthesized by enzyme catalysis, opening a large array of possibilities for this important class of biomass-derived building blocks Because these polymers are potential biomaterials the total absence of conventional Lewis acid catalyst residues represents a major Improvement in the toxicity of the material; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq; FAPESP[2005/02855-7]; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Biocatalysis Center at NYU-POLY; Biocatalysis Center at NYU-POLY

Reações de transferência de acila em microemulsões água/óleo: hidrólise de benzoatos de fenila catalisada pelo ânion o-iodosobenzoato.; Acyl-transfer reactions in water/oil microemulsions: phenyl benzoates hydrolysis catalysed by the o-iodosobenzoate anion.

Bazito, Reinaldo Camino
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 25/07/1997 Português
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Foi estudado o mecanismo da hidrólise de benzoatos de fenila substituídos catalisada pela 1-oxi-1,2-benziodoxol-3(1H)-ona (ânion o-iodosobenzoato, IBA-), em microemulsão água/óleo (mE A/O) de cloreto de benzil-hexadecil-dimetil-amônio (CBzCl) em benzeno. Duas séries de ésteres foram utilizadas: 4-X-benzoatos de 4-nitrofenila (X= NO2, CN, Cl e H) e 4-nitrobenzoatos de Y-fenila (Y= 4-NO2, 3-NO2, 4-CN, 3-CN, 4-Cl e H). Os resultados de IV (detecção do intermediário 1-(4-X-benzoiloxi)-1,2-benziodoxol-3(1H)-ona, benzoil-IBA), ausência de catálise pelo grupo abandonador, e efeito isotópico cinético de solvente inverso mostraram que a catálise pelo IBA- é nucleofílica, ocorrendo em duas etapas: - ataque do IBA- sobre o éster produzindo o intermediário benzoil-IBA e o fenol correspondente; - hidrólise desse intermediário formando os produtos finais da reação, ácido benzóico substituído e IBA-. A intensa absorção do solvente (benzeno) no UV impediu a observação do intermediário benzoil-IBA, por isso somente a primeira etapa da reação (ataque do IBA- sobre o éster, formando o intermediário benzoil-IBA) foi estudada nesta Dissertação. A natureza da etapa lenta da reação, o ataque do IBA- para formar o intermediário tetraédrico éster-IBA...

Síntese e modificação superficial do TiO2 visando aumentar a eficiência do processo de fotocatálise heterogênea no tratamento de compostos fenólicos; Synthesis and superficial modification of TiO2 aiming to improve the efficiency of heterogeneous photocatalysis process on phenolic compounds treatment

Hewer, Thiago Lewis Reis
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 28/11/2005 Português
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Este trabalho de mestrado descreve a avaliação do emprego do TiO2, obtido por diferentes rotas de sínteses e do TiO2 P25 ambos modificados superficialmente com prata e cobre, como catalisador no processo de fotocátalise heterogênea visando a degradação de fenol e efluentes industriais, especificamente efluentes fenólicos. A deterioração dos recursos hídricos é uma das maiores preocupações da sociedade moderna. Dentre os principais responsáveis pela diminuição sistemática na qualidade dos sistemas aquáticos, destacam-se os compostos orgânicos poluentes presentes em efluentes industriais. Dentre esta vasta gama de compostos, os fenóis mostram-se especialmente deletérios ao meio ambiente, devido a sua elevada toxicidade, tendência a bioacumulação e grandes quantidades em que são gerados pelos mais diversos tipos de atividades produtivas. Este trabalho avaliou a aplicação do TiO2 sintetizado pelos métodos de sol-gel e precipitação homogênea na degradação de fenol e no tratamento de efluentes industriais fenólicos. De um modo geral, após 90 minutos de tratamento estes materiais permitiram uma redução de 90% no teor de fenol e de 30% na concentração de carbono orgânico total. Estes resultados são superiores aos obtidos empregando-se TiO¬2 P25 (material comercial mais utilizado neste tipo de processo). A aplicação destes materiais sintetizados na remediação de uma matriz real complexa (efluente industrial) também mostrou uma melhoria na redução de fenol (cerca de 16%) em comparação ao catalisador comercial. Além da síntese do TiO2...

Síntese de polímeros helicoidais para o reconhecimento de moléculas quirais e catálise assimétrica; Synthesis of helical polymers for recognation of chiral molecules and assymetric catalysis

Takata, Leandro Mitsuo Shimura
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 06/03/2015 Português
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Os estudos desenvolvidos nessa tese tiveram como objetivo principal o desenvolvimento de materiais que pudessem ser aplicados tanto para o reconhecimento de moléculas quirais quanto para catálise. A estrutura sugerida baseou-se em nanopartículas de ouro funcionalizadas com polímeros helicoidais ligados à superfície do metal através de um átomo de telúrio. O trabalho foi desenvolvido em quatro etapas, consistindo a primeira dos estudos de funcionalização de nanopartículas de ouro com os organoditeluretos, onde foi observado que a presença de diferentes grupos funcionais não interfere no processo. A segunda etapa foi a investigação da tolerância do catalisador de ródio, necessário para a polimerização, à presença do átomo de telúrio. Os resultados mostraram que a atividade do catalisador é inibida na presença do ditelureto, contudo, o mesmo não ocorre quando o composto está ligado à superfície da nanopartícula. A terceira e quarta etapas foram o desenvolvimento de polímeros helicoidais baseados em estruturas poli(fenilacetilênica)s, sendo uma delas desenvolvida no laboratório do Prof. Alcindo Dos Santos (terceira etapa - Brasil) e a outra no laboratório do Prof. Eiji Yashima (quarta etapa - Japão). Nos estudos da terceira etapa foi dado foco a preparação de um polímero que pudesse ser aplicado tanto para o reconhecimento de moléculas quirais quanto para catálise assimétrica...

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Silva, Rafael G.; Pereira, José H.; Canduri, Fernanda; De Azevedo Jr., Walter F.; Basso, Luiz A.; Santos, Diógenes S.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 49-58
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Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

Synthesis and characterization of 3-[(thiourea)-propyl]-functionalized silica gel and its application in adsorption and catalysis

Vieira, Eduardo Guimarães; Soares, Isaac Vaz; Da Silva, Niléia Cristina; Perujo, Sérgio Daniel; Do Carmo, Devaney Ribeiro; Dias Filho, Newton Luiz
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 1933-1943
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The objective of this research was the preparation of a silica gel functionalized successively with 3-chloropropyltrimethoxysilane (SG-PrCl) and thiourea (SG-Pr-THIO), and its application in adsorption and catalysis. The materials were characterized by 13C and 29Si NMR, FTIR, scanning electron micrographs (SEM), analysis of nitrogen and elemental analysis. Aiming at its application in adsorption, the [3-(thiourea)-propyl] silica gel (SG-Pr-THIO) was tested as an adsorbent for transition-metal ions using a batchwise process. The organofunctionalized surface showed the ability to adsorb the metal ions Cd(ii), Cu(ii), Ni(ii), Pb(ii) and Co(ii) from water, ethanol and acetone. The adsorption isotherms were fitted by Langmuir, Freundlich, Temkin and Dubinin-Radushkevich (D-R) models. The kinetics of adsorption of metals were performed using three models such as pseudo-first order, pseudo-second order and Elovich. The Langmuir and pseudo-first order models were the most appropriate to describe the adsorption and kinetic data, respectively. With the purpose of application in catalysis, the SG-Pr-THIO was reacted with a Mo(ii) organometallic complex, forming the new material SG-Pr-THIO-Mo. Only a few works in the literature have reported this type of reaction...

Extending the kinetic solution of the classic Michaelis-Menten model of enzyme action

BISPO, Jose Ailton Conceicao; BONAFE, Carlos Francisco Sampaio; SOUZA, Volnei Brito de; SILVA, Joao Batista de Almeida e; CARVALHO, Giovani Brandao Mafra de
Fonte: SPRINGER Publicador: SPRINGER
Tipo: Artigo de Revista Científica
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The principal aim of studies of enzyme-mediated reactions has been to provide comparative and quantitative information on enzyme-catalyzed reactions under distinct conditions. The classic Michaelis-Menten model (Biochem Zeit 49:333, 1913) for enzyme kinetic has been widely used to determine important parameters involved in enzyme catalysis, particularly the Michaelis-Menten constant (K (M) ) and the maximum velocity of reaction (V (max) ). Subsequently, a detailed treatment of the mechanisms of enzyme catalysis was undertaken by Briggs-Haldane (Biochem J 19:338, 1925). These authors proposed the steady-state treatment, since its applicability was constrained to this condition. The present work describes an extending solution of the Michaelis-Menten model without the need for such a steady-state restriction. We provide the first analysis of all of the individual reaction constants calculated analytically. Using this approach, it is possible to accurately predict the results under new experimental conditions and to characterize and optimize industrial processes in the fields of chemical and food engineering, pharmaceuticals and biotechnology.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biodiesel production from vegetable frying oil and ethanol using enzymatic catalysis

Paulo Ferreira; Helena Sousa; Nídia Caetano
Fonte: Universidade do Porto Publicador: Universidade do Porto
Tipo: Artigo de Revista Científica
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The aim of this work was the evaluation and optimization of conditions for Fatty Acid Ethyl Ester (FAEE) production. The influence of variables such as catalyst (NaOH, Lipozyme RM IM, Lipozyme TL IM, Novozym 435), alcohol (methanol, ethanol 96% v/v, ethanol 70% v/v), reaction temperature (35 38°C), oil: alcohol molar ratio (1:3; 1:9; 1:18) and reaction time (8 and 24h) was studied.Comparing alkaline homogeneous with enzymatic catalysis, we could observe that the former one produced a higher yield and lower selectivity, in addition to minor problems related to corrosion of the equipment used in the process.Enzymatic catalysis also presents another benefit, which is the fact that it allows no side reactions producing byproducts, therefore reducing the costs on subsequent purification. In this context, enzymatic catalysts have been studied as an alternative to FAEE production, so that separation processes can be simplified and associated costs reduced.Experimental runs when Lipozyme TL IM was used lead to the best results. Yields ranging from 21 to 60% where achieved in 8 hours reaction time. However, the highest yield (90%) was obtained using 96% v/v ethanol, an alcohol : oil ratio of 1:3, 24 hours reaction time and temperature range of 35-38ºC.

Sucrose Inversion An Experiment on Heterogeneous Catalysis

Adélio Mendes; Fernão D. Magalhães; Luis M. Madeira
Fonte: Universidade do Porto Publicador: Universidade do Porto
Tipo: Artigo de Revista Científica
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llustration of heterogeneous catalysis concepts in laboratory courses is not usually simple or economical. For our undergraduate senior lab course we have developed an environmentally friendly experiment dealing with several aspects of heterogeneous catalysis, having in mind the use of readily available and relatively inexpensive equipment, and chemicals on a compact setup, which students can safely operate. The experiment deals with the acid-catalyzed sucrose inversion, performed in packed bed chemical reactors, where the catalyst is a cation-exchange resin in the H + form. An additional reactor is included for illustrating an enzyme-catalyzed system. The conversion achieved is determined using the Flow Injection Analysis technique.

Síntese e aplicação de novos organocatalisadores de alcalóides de Cinchona em catálise assimétrica

Barrulas, Pedro Miguel Cambeiro
Fonte: Universidade de Évora Publicador: Universidade de Évora
Tipo: Tese de Doutorado
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Esta tese descreve o projeto, síntese e caracterização de novos derivados de cinchonidina e teve como objetivo central a sua aplicação como organocatalisadores enantiosseletivos em reações catalíticas assimétricas. Os alcalóides de Cinchona modificados com êxito através de metodologias simples, puderam ser agrupados em três famílias distintas, nomeadamente em 1,2,3-triazóis, aminoácidos híbridos e piridinacarboxamidas derivadas da cinchonidina. A capacidade organocatalítica destes compostos foi então estudada em reações assimétricas de Michael, de Biginelli, aldólicas e em hidrossililações de cetiminas, revelando-se um sucesso para os alcalóides modificados com aminoácidos e piridinacarboxamidas. Com estas estruturas foram alcançados bons rendimentos reacionais e elevadas enantiosseletividades em reações aldólicas e em hidrossililações de cetiminas, respetivamente. Destaca-se ainda a potencial e desejada aplicação destas estruturas na síntese enantiosseletiva de compostos biologicamente ativos num futuro próximo. Os bons resultados permitiram o estudo e aplicação dos novos organocatalisadores em diferentes suportes sólidos e a respetiva aplicação em catálise heterogénea assimétrica; Abstract: Synthesis and Application of Novel Organocatalysts Based on Cinchona Alkaloids for Asymmetric Catalysis This thesis describes the design...

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

Silva, Teresa Santos; Ferroni, Felix; Thapper, Anders; Marangon, Jacopo; Moura, Isabel; Moura, José J. G.; Rizzi, Alberto C.; González, Pablo J.; Romão, Maria J.; Brondino, Carlos D.
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448r; Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase(XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V)EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme...

Ab initio investigation of the kinetics and mechanism of the neutral hydrolysis of formamide in aqueous solution

Almerindo,Gizelle I.; Pliego Jr.,Josefredo R.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2007 Português
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The neutral hydrolysis of formamide in aqueous solution involving up to four explicit solvent molecules was theoretically investigated using high level ab initio methods and including the bulk solvent effect through the Polarizable Continuum Model (PCM). We have analyzed cyclic transition states structures involving bifunctional catalysis and a general base catalysis structure. Both a stepwise mechanism with tetrahedral intermediate formation and a concerted mechanism were investigated. Our calculations at CCSD(T)/6-311+G(2df,2p)//MP2/6-31G(d) level predict an observable activation free energy barrier of 48.7 kcal mol-1, corresponding to a stepwise water catalyzed mechanism with two water molecules into the transition state. Liquid phase geometry optimization was also performed, but the effect on the activation free energy is modest. Tests with density functional theory were carried out. The B3LYP/6-31G(d) calculation underestimates the barrier by 13 kcal mol-1, whereas the B3LYP/6-311+G(2df,2p) method predicts an accurate barrier. The present study raises important questions about the reliability of the experimental activation free energy of 31.0 kcal mol-1 and suggests that the neutral hydrolysis of formamide does not take place at all.

Aqueous Micelles as Catalytic Nanoreactors for Dephosphorylation Reactions

Wanderlind,Eduardo H.; Orth,Elisa S.; Medeiros,Michelle; Santos,Deise M. P. O.; Westphal,Eduard; Gallardo,Hugo; Fiedler,Haidi D.; Nome,Faruk
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2014 Português
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The reaction of a new imidazole derivative, 3,5-bis((1H-imidazol-1-yl)methyl)aniline (BIm), and the model triester diethyl 2,4-dinitrophenyl phosphate (DEDNPP), which proceeds via a SN2(P) mechanism, is substantially enhanced in the presence of either cationic cetyltrimethylammonium bromide (CTAB) or anionic sodium dodecyl sulfate (SDS). This unusual micellar effect illustrates the use of the hydrophobic character of the reactants to enhance micellar catalysis. This is an important and useful guideline in planning mimics for biological reactions, because improving incorporation will increase the quality of the biomimetic systems, and both cationic or anionic surfactants will act as homogeneous nanoreactors concentrating the neutral, hydrophobic reactants.

Aspects on the catalysis of lipase from porcine pancreas (type VI-s) in aqueous media: development of ion-pairs

Kokkinou,Marina; Theodorou,Leonidas G.; Papamichael,Emmanuel M.
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2012 Português
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This article reports a first contribution for the elucidation of catalytic mechanism of Lipase from porcine pancreas, type VI-s (PPL), in hydrolyzing an ester substrate in aqueous media. The conclusions were based on the pH-profiles of Michaelis-Menten parameters k cat/Km, k cat and Km, as well as on the absolute temperature profile of k cat/Km, obtained during the hydrolysis of p-nitrophenyl laurate by PPL. It was found that (a) PPL performs catalysis by means of ion pairs formed either as Ser152-Ο-/His263-Im+H and/or Carbonyl-Ο-/His263-Im+H, (b) the parameter k cat/Km equals to k1 and thus ES is formed and destroyed in the course of a series of consecutive reactions governed by the dynamic constant K S = k2/k1, and (c) the hydrolysis of substrate is assisted by a hydrogen bond developed between deprotonated Asp176 and the positively charged imidazole of His263 across a pKa-value 3.85, necessary for efficient catalysis.

Asparagine 229 in thymidylate synthase contributes to, but is not essential for, catalysis.

Liu, L; Santi, D V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/09/1993 Português
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The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229...

Pause and rotation of F1-ATPase during catalysis

Hirono-Hara, Yoko; Noji, Hiroyuki; Nishiura, Masaya; Muneyuki, Eiro; Hara, Kiyotaka Y.; Yasuda, Ryohei; Kinosita, Kazuhiko; Yoshida, Masasuke
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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F1-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α3β3 ring. Here, we show that the rotation of F1-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F1-ATPase previously known as the ADP-Mg inhibited form in which F1-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.

A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis

Heath, Richard J.; Rock, Charles O.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/1998 Português
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Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane...

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler, Erik; Thorell, Stina; Sandalova, Tatyana; Golbik, Ralph; König, Stephan; Schneider, Gunter
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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Kinetic and spectroscopic data indicated that addition of the donor substrate hydroxypyruvate to the thiamin diphosphate (ThDP)-dependent enzyme transketolase (TK) led to the accumulation of the α-carbanion/enamine of (α,β-dihydroxyethyl) ThDP, the key reaction intermediate in enzymatic thiamin catalysis. The three-dimensional structure of this intermediate trapped in the active site of yeast TK was determined to 1.9-Å resolution by using cryocrystallography. The electron density suggests a planar α-carbanion/enamine intermediate having the E-configuration. The reaction intermediate is firmly held in place through direct hydrogen bonds to His-103 and His-481 and an indirect hydrogen bond via a water molecule to His-69. The 4-NH2 group of the amino-pyrimidine ring of ThDP is within 3 Å distance to the α-hydroxy oxygen atom of the dihydroxyethyl moiety but at an angle unfavorable for a strong hydrogen bond. No structural changes occur in TK on formation of the reaction intermediate, suggesting that the active site is poised for catalysis and conformational changes during the enzyme reaction are not very likely. The intermediate is present with high occupancy in both active sites, arguing against previous proposals of half-of-the-sites reactivity in yeast TK.

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Bosco, Daryl A.; Eisenmesser, Elan Z.; Pochapsky, Susan; Sundquist, Wesley I.; Kern, Dorothee
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89–Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CAN). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CAN has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CAN but also catalyzes efficiently the cis/trans isomerization of the Gly-89–Pro-90 peptide bond. In addition, conformational changes in CAN distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.