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Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

CHELESKI, Juliana; FREITAS, Renato F.; WIGGERS, Helton Jose; ROCHA, Josmar R.; ARAUJO, Ana Paula Ulian de; MONTANARI, Carlos A.
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
56.04%
Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M...

Caracterização cinética da (Na, K)-ATPase da fração microsomal do tecido branquial do ermitão intertidal Clibanarius vittatus; Kinetic characterization of gill microsomal (Na+,K+)- ATPase from intertidal hermit crab Clibanarius vittatus

Sivieri, Rubia Regina Gonçalves
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 23/03/2007 PT
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66.15%
A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial do sistema de regulação iônica e osmótica e de excreção ativa de NH4 +. Dessa forma, a caracterização cinética da (Na+,K+)-ATPase branquial de Clibanarius vittatus pode fornecer dados importantes para a compreensão dos mecanismos bioquímicos desses processos. Nesse sentido, foram realizados ensaios cinéticos com a fração microsomal obtida do tecido branquial de C. vittatus. Os dados obtidos revelaram a presença de um único pico com atividade ATPase. Os dados obtidos a partir da hidrólise do substrato ATP revelaram a presença de dois sítios, um de alta afinidade que apresenta interação sítio-sítio (nH=1,9; K0,5= 63,8 ± 2,9 nM e V= 19,1 ± 0,8 U/mg) e outro de baixa afinidade, com características Michaelianas (nH=1,0; KM= 44,1 ± 2,6 mM e VM= 123,5 ± 6,1 U/mg). Além disso, foi observada interação do tipo sítio-sítio (nH= 1,8) para estimulação dos íons magnésio (V= 132,0 ± 5,3 U/mg e K0,5= 0,36 ± 0,02 mM). Por outro lado, as interações dos íons sódio (K0,5= 7,4 ± 0,4 mM), potássio (K0,5= 1,51 ± 0,05 mM) e amônio (K0,5= 4,5 ± 0,2 mM) obedeceram a uma cinética Michaeliana. Os dados obtidos mostraram que em condições saturantes de íons potássio...

Purificação e caracterização de uma carboxipeptidase e de uma dipeptidase da larva de Tenebrio molitor (Coleoptera); Purification and characterization of a carboxypeptidase and a dipeptidase from Tenebrio molitor (Coleoptera) larvae

Oliveira, Érica Moreira de
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 07/08/2008 PT
Relevância na Pesquisa
45.98%
Devido aos problemas, ambientais e à população humana, causados pelos inseticidas químicos, novas investigações para o controle de insetos tornaram-se necessárias. Para isto, um maior conhecimento sobre a fisiologia digestiva dos insetos torna-se essencial, visto que o intestino é uma interface, grande e relativamente desprotegida, entre o inseto e o seu ambiente. Neste contexto, nosso trabalho envolve a purificação e caracterização de uma dipeptidase e de uma carboxipeptidase digestivas de T. molitor. Estas enzimas, compreendem as classes de enzimas digestivas de insetos menos estudadas. O estudo de distribuição das atividades de dipeptidase e carboxipeptidase nas diferentes regiões do intestino médio de larvas de T. molitor mostrou que essas enzimas encontram-se majoritariamente no conteúdo luminal. Para a purificação da dipeptidase e carboxipeptidase foram utilizadas cromatografias de troca iônica e filtração em gel. A carboxipeptidase purificada era muito instável para estudos mais detalhados. O estudo dos parâmetros cinéticos mostrou que a dipeptidase digestiva de T. molitor possui massa molecular de 38,6 kDa, pH ótimo 7,4, baixa solubilidade a fenantrolina e parece preferir dipeptídeos com cadeia lateral volumosa na posição P1. Devido a essas propriedades...

Caracterização cinética da (Na+, K+)-ATPase de animais juvenis e adultos durante a ontogenia do camarão de água doce M. amazonicum; Kinetic characterization of (Na +, K +)-ATPase of juvenile and adult animals during ontogeny of the freshwater shrimp M. amazonicum.

Bezerra, Thaís Milena de Souza
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 28/05/2010 PT
Relevância na Pesquisa
45.92%
No presente trabalho foram estudadas a caracterização cinética e as propriedades bioquímicas da (Na+, K+)-ATPase branquial dos estágios ontogenéticos juvenil e adulto do camarão de água doce M. amazonicum. Em ambos os estágios de desenvolvimento foi expressa uma (Na+, K+)-ATPase com peso molecular de 105 kDa. A estimulação da atividade (Na+, K+)-ATPase de animais adultos pelo ATP apresentou comportamento michaeliano (V=181,8±77,83 U mg-1; KM=0,11±0,01 mmol L-1). Entretanto a estimulação da atividade (Na+, K+)-ATPase pelo Mg2+ (V=174,7±74,76 U mg-1; K0,5=0,27±0,05 mmol L-1; n= 2,5), Na+ (V=175,5±75,12 U mg-1; K0,5=4,73±0,81 mmol L-1; n= 1,9), K+ (V=171,2±73,28 U mg-1; K0,5=1,00±0,17 mmol L-1; n=1,2) e NH4+ (V=194,2±63,34 U mg-1; K0,5= 4,76±2,15 mmol L-1; n=1,9) ocorreu segundo cinética cooperativa. Nos animais juvenis, a modulação da atividade (Na+, K+)-ATPase pelo ATP, também apresentou comportamento michaeliano (V=189,52±32,45 U mg-1; KM= 0,14±0,02 mmol L-1). O mesmo foi observado para o K+ (V=178,56±30,58 U mg-1; KM= 1,30±0,22 mmol L-1) e o NH4+ (V=205,91±35,26 U mg-1; KM=1,88±0,32 mmol L-1). Para o Mg2+ (V=168,35±28,83 U mg-1; K0,5=0,51±0,09 mmol L-1; n=3,5) e o Na+ (V=165,48±28,33 U mg-1; K0...

Adaptção de linhagens celulares humanas para crescimento em suspensão e meios de cultura livres de soro fetal bovino; Serum-free suspension adaptation of human cell lines

Biaggio, Rafael Tagé
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 28/03/2014 PT
Relevância na Pesquisa
45.92%
Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8...

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

Moreira, Ariele C.; Ferreira, Douglas; Almeida, Fernando G. de; Rodrigues-Filho, Edson; Fernandes, Joao B.; Silva, Maria F. G. F.; Vieira, Paulo C.; Pagnocca, Fernando C.; Souza, Dulce Helena F.
Fonte: Humana Press Inc Publicador: Humana Press Inc
Tipo: Artigo de Revista Científica Formato: 694-704
ENG
Relevância na Pesquisa
45.96%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Processo FAPESP: 11/21955-3; In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 A degrees C, optimum pH between 8.5 and 10.5, and Km = 14.7 A +/- 7.6 mg mL(-1). Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 A degrees C, and Km = 2.2 A +/- 0.5 mg mL(-1). XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 A degrees C) and pH (4.0), as well as the kinetic parameters (Km = 7.4 A +/- 2.0 mg mL(-1)) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.

Isolation and characterization of a newly isolated Pseudomonas mutant for protease production

Dutta,Jayati Ray; Banerjee,Rintu
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2006 EN
Relevância na Pesquisa
45.92%
A potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044. A mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34ºC, inoculum volume of 1.0 mL and incubation time of 24 hours. Comparative analysis of the chemical characteristics i.e. assimilation of carbon and nitrogen sources were also carried out. Maximum growth of the mutant strain in 2% gelatin agar plate was obtained in presence of dextrose (2%), maltose (2%), ammonium sulfate (2%) and potassium nitrate (2%) whereas, that of the parent strain was found in sucrose (2%) and ammonium nitrate (2%). The purified proteases from both the strains (parent and mutant) appeared as single homogeneous bands corresponding to 14.4 kDa molecular weight on SDS-PAGE. On studying the kinetic properties of both strains it was observed that the rate of casein hydrolysis was maximum at pH 8.0 and 7.0 and temperatures 45º C and 60º C for the parent and mutant strains respectively. It was also observed that both the extracellular proteases were inhibited by a serine protease inhibitor i.e. PMSF at 2mM concentration.

Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme

Umbreen,Huma; Zia,Muhammad Anjum; Rasul,Samreen
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2013 EN
Relevância na Pesquisa
45.92%
In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL), 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km) were 20 mM, 45.87 U mL-1, 1118.81 s-1 and 55.94 s-1 mM-1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness.

The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B.

Walker, B; Cullen, B M; Kay, G; Halliday, I M; McGinty, A; Nelson, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/04/1992 EN
Relevância na Pesquisa
45.92%
In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase...

The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.

Kay, G; Bailie, J R; Halliday, I M; Nelson, J; Walker, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/04/1992 EN
Relevância na Pesquisa
45.92%
The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated...

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: in vivo kinetic characterization of 2,3-bisphosphoglycerate synthase/phosphatase using 13C and 31P NMR.

Mulquiney, P J; Bubb, W A; Kuchel, P W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/09/1999 EN
Relevância na Pesquisa
46.04%
This is the first in a series of three papers [see also Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594; Mulquiney and Kuchel (1999) Biochem. J. 342, 595-602] that present a detailed mathematical model of erythrocyte metabolism which explains the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is a modulator of haemoglobin oxygen affinity and hence plays an important role in blood oxygen transport and delivery. This paper presents an in vivo kinetic characterization of 2,3-BPG synthase/phosphatase (BPGS/P), the enzyme that catalyses both the synthesis and degradation of 2,3-BPG. Much previous work had indicated that the behaviour of this enzyme in vitro is markedly different from that in vivo. (13)C and (31)P NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-(13)C]glucose. Simulations of the experimental time courses were then made. By iteratively changing the parameters of the BPGS/P part of the model until a good match between the NMR-derived data and simulations were achieved, it was possible to characterize BPGS/P kinetically in vivo. This work revealed that: (1) the pH-dependence of the synthase activity results largely from a strong co-operative inhibition of the synthase activity by protons; (2) 3-phosphoglycerate and 2-phosphoglycerate are much weaker inhibitors of 2...

Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates

Heyes, Derren J.; Kruk, Jerzy; Hunter, C. Neil
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46.04%
The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four ‘dark’ steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme...

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 02/03/1996 EN
Relevância na Pesquisa
45.92%
Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy- chain phosphorylation, and (d) ADP release is fast. Thus, during steady- state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily...

Kinetic Characterization of the WalRKSpn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalKSpn (VicK) Phosphatase Activity on Its PAS Domain▿ †

Gutu, Alina D.; Wayne, Kyle J.; Sham, Lok-To; Winkler, Malcolm E.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46%
The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalRSpn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalRSpn is phosphorylated by the WalKSpn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalKSpn signal transduction, we performed a kinetic characterization of the WalRKSpn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalKSpn. Consequently, these analyses were performed using two truncated versions of WalKSpn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalKSpn were similar [Km(ATP) ≈ 37 μM; kcat ≈ 0.10 min−1] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalKSpn∼P were also similar in the phosphoryltransfer reaction to full-length WalRSpn. In contrast...

A Pathogenic Linked Mutation in the Catalytic Core of Human Cystathionine β-synthase Disrupts Allosteric Regulation and Allows Kinetic Characterization of a Full-length Dimer†

Sen, Suvajit; Banerjee, Ruma
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46.04%
Cystathionine β-synthase catalyzes the condensation of serine and homocysteine to yield cystathionine and is the single most common locus of mutations associated with homocystinuria. In this study, we have examined the kinetic consequences of a pair of linked patient mutations, P78R/K102N, that are housed in the catalytic core of the protein and compared it to the effects of the corresponding single mutations. The P78R mutation affords purification of a mixture of higher order oligomers, P78R-I, which resembles the mixed quaternary state associated with wild type enzyme. However, unlike wild type enzyme, P78R-I converts over time to P78R-II, which exists predominantly, as a full-length dimer. The specific activities of the K102N, P78R-I and P78R-II mutants in the absence of AdoMet are ~3-, 9- and 3-fold lower than of wild-type enzyme and are stimulated 2.9-, 2.5- and 1.4-fold respectively by AdoMet. However, when linked, the specific activity of the resulting double mutant is comparable to that of wild-type enzyme but it is unresponsive to AdoMet, revealing that interactions between the two sites modulate the phenotype of the enzyme. Steady-state kinetic analysis for the double mutant reveals a sigmoidal dependence on homocysteine that is not observed with wild-type enzyme...

Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at threonine668

De, Soumya; Greenwood, Alexander I.; Rogals, Monique J.; Kovrigin, Evgenii L.; Lu, Kun Ping; Nicholson, Linda K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46%
Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into timescales relevant for cellular signaling. Here we have combined NMR lineshape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ~22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore...

Kinetic Characterization of Exonuclease-Deficient Staphylococcus aureus PolC, a C-family Replicative DNA Polymerase

Lahiri, Indrajit; Mukherjee, Purba; Pata, Janice D.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/05/2013 EN
Relevância na Pesquisa
46%
PolC is the C-family replicative polymerase in low G+C content Gram-positive bacteria. To date several structures of C-family polymerases have been reported, including a high resolution crystal structure of a ternary complex of PolC with DNA and incoming deoxynucleoside triphosphate (dNTP). However, kinetic information needed to understand the enzymatic mechanism of C-family polymerases is limited. For this study we have performed a detailed steady-state and pre-steady-state kinetic characterization of correct dNTP incorporation by PolC from the Gram-positive pathogen Staphylococcus aureus, using a construct lacking both the non-conserved N-terminal domain and the 3′–5′ exonuclease domain (Sau-PolC-ΔNΔExo). We find that Sau-PolC-ΔNΔExo has a very fast catalytic rate (kpol 330 s−1) but also dissociates from DNA rapidly (koff ∼150 s−1), which explains the low processivity of PolC in the absence of sliding clamp processivity factor. Although Sau-PolC-ΔNΔExo follows the overall enzymatic pathway defined for other polymerases, some significant differences exist. The most striking feature is that the nucleotidyl transfer reaction for Sau-PolC-ΔNΔExo is reversible and is in equilibrium with dNTP binding. Simulation of the reaction pathway suggests that rate of pyrophosphate release...

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Glenn, Katie; Ingram-Smith, Cheryl; Smith, Kerry S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2014 EN
Relevância na Pesquisa
46.06%
Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC50) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean)...

Kinetic study of the co-firing of bagasse-sludge blends

Torquato, Lilian M.; Braz, Carlos E. M.; Ribeiro, Clovis A.; Capela, Jorge M. V.; Crespi, Marisa S.
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica Formato: 499-507
ENG
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Thermogravimetry was used for the thermal and kinetic characterization of waste biomass as bagasse and sewage sludge and their blends, during the co-firing process for power generation. The kinetic evaluation was performed by a linear integral isoconversional method, applying different conditions of heating. In higher heating rates, kinetic process of combustion of all tested biomasses showed the lower values and the lower changes in activation energy up to 70 % for the conversion of all samples. Above this point, there was a continuous increase in this parameter for bagasse and the bagasse-sludge blends up to the end of the processes. However, in the case of sludge, there was a continuous decrease in energy, which suggests the occurrence of catalytic reactions. The results showed that under low heating rates, the sludge-bagasse blends exhibit profiles of kinetic decomposition very similar to bagasse, regardless the proportion of sludge employed, which may be due the contribution of the high amount of volatile organic materials present in bagasse (30 % higher than the sludge), which increase the reactivity of the sample during the devolatilization process. On the other hand...

Structural and Kinetic Characterization of LpxK, the Tetraacyldisaccharide-1- Phosphate Kinase of Lipid A Biosynthesis

Emptage, Ryan Paul
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2013
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Lipopolysaccharide, the physical barrier that protects Gram-negative bacteria from various antibiotics and environmental stressors, is anchored to the outer membrane by the phosphorylated, acylated disaccharide of glucosamine known as lipid A. Besides being necessary for the viability of most Gram-negative bacteria, lipid A interacts directly with specific mammalian immune cell receptors, causing an inflammatory response that can result in septic shock. The lipid A biosynthetic pathway contains nine enzymatic steps, the sixth being the phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) precursor to form lipid IVA by the inner membrane-bound kinase LpxK, a divergent member of the P-loop containing nucleotide triphosphate hydrolase superfamily. LpxK is the only known P-loop kinase to act on a lipid at the membrane interface.

We report herein multiple crystal structures of Aquifex aeolicus LpxK in apo as well as ATP, ADP/Mg2+, AMP-PCP, and chloride-bound forms. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other P-loop kinase family members, is responsible for catalysis at the P-loop and positioning of the DSMP substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain...