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Enzyme kinetics

Buki Kwon, Palinda Ruvan Munashingha, Yong-Keol Shin, Chul-Hwan Lee, Bing Li, Yeon-Soo Seo
Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse posttranslational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes...
October 19, 2016: FEBS Journal
Ross D Blundell, Simon J Williams, Samantha D M Arras, Jessica L Chitty, Kirsten L Blake, Daniel J Ericsson, Nidhi Tibrewal, Jurgen Rohr, Y Q Andre E Koh, Ulrike Kappler, Avril A B Robertson, Mark S Butler, Matthew A Cooper, Bostjan Kobe, James A Fraser
Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C...
September 9, 2016: ACS Infectious Diseases
Scott J Hughes, Leanne Barnard, Katayoun Mottaghi, Wolfram Tempel, Tetyana Antoshchenko, Bum Soo Hong, Abdellah Allali-Hassani, David Smil, Masoud Vedadi, Erick Strauss, Hee-Won Park
The potent antistaphylococcal activity of N-substituted pantothenamides (PanAms) has been shown to at least partially be due to the inhibition of Staphylococcus aureus's atypical type II pantothenate kinase (SaPanKII), the first enzyme of coenzyme A biosynthesis. This mechanism of action follows from SaPanKII having a binding mode for PanAms that is distinct from those of other PanKs. To dissect the molecular interactions responsible for PanAm inhibitory activity, we conducted a mini SAR study in tandem with the cocrystallization of SaPanKII with two classic PanAms (N5-Pan and N7-Pan), culminating in the synthesis and characterization of two new PanAms, N-Pip-PanAm and MeO-N5-PanAm...
September 9, 2016: ACS Infectious Diseases
Aníbal M Reyes, Diego S Vazquez, Ari Zeida, Martín Hugo, M Dolores Piñeyro, María Inés De Armas, Darío Estrin, Rafael Radi, Javier Santos, Madia Trujillo
Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M...
October 14, 2016: Free Radical Biology & Medicine
Bosko M Stojanovski, Leonid Breydo, Vladimir N Uversky, Gloria C Ferreira
In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline molten globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS). The stability of the molten globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10...
October 14, 2016: Biochemical and Biophysical Research Communications
Carlos Gueto-Tettay, Joshua Zuchniarz, Yeyson Fortich-Seca, Luis Roberto Gueto-Tettay, Juan Carlos Drosos-Ramirez
BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aβ-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme...
October 7, 2016: Journal of Molecular Graphics & Modelling
Jaclyn A Adkins, Eka Noviana, Charles S Henry
An electrochemical paper-based analytical device (ePAD) was developed for quasi-steady flow detection at microwire electrodes, for the first time. The device implements a fan shaped geometry connected to an analysis channel whereby solution is pulled from an inlet, through a channel, and into the steadily increasing capillary network of the fan. The network counteracts the decrease in solution flow rate associated with increasing viscosity within the channel, generating quasi-steady flow within the analysis channel...
October 17, 2016: Analytical Chemistry
Salette Martinez, Robert P Hausinger
The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. This enzyme is reported to simultaneously catalyze the conversion of 2OG into ethylene plus three CO2 and the Cδ hydroxylation of L-arginine (L-Arg) while oxidatively decarboxylating 2OG to form succinate and carbon dioxide. A new plasmid construct for expression in recombinant Escherichia coli cells allowed for the purification of large amounts of EFE with greater activity than previously recorded...
October 17, 2016: Biochemistry
Ricardo da Silva Raposo, Vamilton Alvares Santarém, Yslla Fernanda Fitz Balo Merigueti, Guita Rubinsky-Elefant, Letícia Maria de Lima Cerazo, Ludimilla Pereira, Bianca Pelegi Zampieri, Aristeu Vieira da Silva, Cecília Braga Laposy
Toxocariasis is a geohelminth zoonosis with worldwide distribution, mainly transmitted through the ingestion of embryonated eggs of nematodes of the Toxocara genus. The disease can also be transmitted to humans as a result of eating raw or undercooked meat of paratenic hosts, such as chickens. Here, we standardized an enzyme-linked immunosorbent assay (ELISA) for evaluating experimentally the kinetic and avidity index (AI) of IgY in broiler chickens infected with different doses of Toxocara canis eggs (G1:100; G2: 1000; and G3: 5000; n = 12 per group)...
October 13, 2016: Experimental Parasitology
Felix Bäuerle, Agnes Zotter, Gideon Schreiber
With computer-based data-fitting methods becoming a standard tool in biochemistry, progress curve analysis of enzyme kinetics is a feasible, yet seldom used tool. Here we present a versatile Matlab-based tool (PCAT) to analyze catalysis progress curves with three complementary model approaches. The first two models are based on the known closed-form solution for this problem: the first describes the required Lambert W function with an analytical approximation and the second provides a numerical solution of the Lambert W function...
October 15, 2016: Protein Engineering, Design & Selection: PEDS
T G Fonseca, M B Morais, T Rocha, D M S Abessa, M Aureliano, M J Bebianno
Anticancer drugs are designed to inhibit tumor cell proliferation by interacting with DNA and altering cellular growth factors. When released into the waterbodies of municipal and hospital effluents these pharmaceutical compounds may pose a risk to non-target aquatic organisms, due to their mode of action (cytotoxic, genotoxic, mutagenic and teratogenic). The present study aimed to assess the ecotoxicological potential of the alkylating agent cisplatin (CisPt) to the polychaete Nereis diversicolor, at a range of relevant environmental concentrations (i...
October 12, 2016: Science of the Total Environment
Dr T J Lasisi, S T Shittu, C C Meludu, A A Salami
OBJECTIVE: Aim of this study was to investigate the effects of sleep deprivation on salivary factors in rats. DESIGN: Animals were randomly assigned into three groups of 6 animals each as control, total sleep deprivation (TSD) and partial sleep deprivation (PSD) groups. The multiple platform method was used to induce partial and total sleep deprivation for 7days. On the 8th day, stimulated saliva samples were collected for the analysis of salivary lag time, flow rate, salivary amylase activity, immunoglobulin A secretion rate and corticosterone levels using ELISA and standard kinetic enzyme assay...
September 26, 2016: Archives of Oral Biology
Diogo R B Ducatti, Madison A Carroll, David L Jakeman
A phosphorolytic activity has been reported for beta-N-acetylglucosaminidases from glycoside hydrolase family 3 (GH3) giving an interesting explanation for an unusual histidine as catalytic acid/base residue and suggesting that members from this family may be phosphorylases [J. Biol. Chem. 2015, 290, 4887]. Here, we describe the characterization of Hsero1941, a GH3 beta-N-acetylglucosaminidase from the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1. The enzyme has significantly higher activity against pNP-beta-D-GlcNAcp (Km = 0...
September 23, 2016: Carbohydrate Research
Patricie Burda, Terttu Suormala, Dorothea Heuberger, Alexandra Schäfer, Brian Fowler, D Sean Froese, Matthias R Baumgartner
5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression...
October 14, 2016: Journal of Inherited Metabolic Disease
Ali Altıntaş, Kristian Davidsen, Christian Garde, Uffe H Mortensen, J Christian Brasen, Thomas Sams, Christopher T Workman
Although the role of oxidative stress factors and their regulation is well studied, the temporal dynamics of stress recovery is still poorly understood. In particular, measuring the kinetics of stress recovery in the first minutes after acute exposure provides a powerful technique for assessing the role of regulatory proteins or enzymes through the use of mutant backgrounds. This project endeavors to screen the temporal dynamics of intracellular oxidant levels in live cells as a function of gene deletion in the budding yeast, Saccharomyces cerevisiae...
October 11, 2016: Free Radical Biology & Medicine
Chase A Klingaman, Matthew J Wagner, Justin R Brown, John B Klecker, Ethan H Pauley, Colin J Noldner, Jared R Mays
Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development...
October 11, 2016: Analytical Biochemistry
Scott Mazurkewich, Stephen Y K Seah
The 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is the last enzyme of both the gallate and protocatechuate 4,5-cleavage pathways which links aromatic catabolism to central cellular metabolism. The enzyme is a class II, divalent metal dependent, aldolase which is activated in the presence of inorganic phosphate (Pi), increasing its turnover rate >10-fold. This phosphate activation is unique for a class II aldolase. The aldolase pyruvate methyl proton exchange rate, a probe of the general acid half reaction, was increased 300-fold in the presence of 1 mM Pi and the rate enhancement followed saturation kinetics giving rise to a KM of 397 ± 30 μM...
2016: PloS One
Liao Peng, Anchao Feng, Senyang Liu, Meng Huo, Tommy Fang, Ke Wang, Yen Wei, Xiaosong Wang, Jinying Yuan
Potential-stimulated Pickering emulsions, using electrochemical responsive microgels as particle stabilizers, are prepared and used for biocatalysis. The microgels are constructed from the cyclodextrin functionalized 8-arm poly(ethylene glycol) (8A PEG-CD) and ferrocene modified counterparts (8A PEG-Fc) via a CD-Fc host-guest chemistry. Taking advantage of the redox reaction of Fc, the formation and deformation of the microgels and corresponding Pickering emulsions can be reversibly stimulated by external potential, and have been used for the hydrolysis of triacetin and kinetic resolution reaction of (R,S)-1-phenylethanol catalyzed by lipases...
October 14, 2016: ACS Applied Materials & Interfaces
Sowmyalakshmi Venkataraman, Shoba Narayan, Anju Chadha
Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells...
October 14, 2016: Scientific Reports
Andrea von Zadow, Elisabeth Ignatz, Richard Pokorny, Lars-Oliver Essen, Gabriele Klug
Photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions, instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides was until now described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition...
October 14, 2016: FEBS Journal
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