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Enzyme kinetics

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https://www.readbyqxmd.com/read/28934493/ubalai-is-a-monomeric-type-iie-restriction-enzyme
#1
Giedrius Sasnauskas, Giedre Tamulaitiene, Gintautas Tamulaitis, Jelena Calyševa, Migle Laime, Renata Rimšeliene, Arvydas Lubys, Virginijus Siksnys
Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5'-CCWGG-3' sequence...
September 19, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28934415/first-report-of-urease-activity-in-the-novel-systemic-fungal-pathogen-emergomyces-africanus-a-comparison-with-the-neurotrope-cryptococcus-neoformans
#2
Barbra Lerm, Chris Kenyon, Ilan S Schwartz, Heinrich Kroukamp, Riaan de Witt, Nelesh P Govender, G Sybren de Hoog, Alfred Botha
Cryptococcus neoformans is an opportunistic pathogen responsible for the AIDS-defining illness, cryptococcal meningitis. During the disease process, entry of cryptococcal cells into the brain is facilitated by virulence factors that include urease enzyme activity. A novel species of an Emmonsia-like fungus, recently named Emergomyces africanus, was identified as a cause of disseminated mycosis in HIV-infected persons in South Africa. However, in contrast to C. neoformans, the enzymes produced by this fungus, some of which may be involved in pathogenesis, have not been described...
November 1, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28933751/rapid-mechanistic-evaluation-and-parameter-estimation-of-putative-inhibitors-in-a-single-step-progress-curve-analysis-the-case-of-horse-butyrylcholinesterase
#3
Jure Stojan
Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing according to modified reaction mechanisms...
July 26, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28933564/%C3%AE-glucosidase-inhibition-by-flavonoids-an-in-vitro-and-in-silico-structure-activity-relationship-study
#4
Carina Proença, Marisa Freitas, Daniela Ribeiro, Eduardo F T Oliveira, Joana L C Sousa, Sara M Tomé, Maria J Ramos, Artur M S Silva, Pedro A Fernandes, Eduarda Fernandes
α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes mellitus. As flavonoids are promising modulators of this enzyme's activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure-activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds...
December 2017: Journal of Enzyme Inhibition and Medicinal Chemistry
https://www.readbyqxmd.com/read/28932939/the-thumb-domain-is-not-essential-for-the-catalytic-action-of-holama-dna-polymerase
#5
Angela Gala Morena Gatius, Fabrizio Dal Piaz, Alejandro Hochkoeppler
A structural and kinetic characterization of a fragment of the HoLaMa DNA polymerase is presented here. In particular, a truncated form of HoLaMa, devoid of a consistent portion of the thumb domain, was isolated and purified. This HoLaMa fragment, denoted as ΔNter-HoLaMa, is surprisingly competent in catalyzing DNA extension, albeit featuring a kcat one order of magnitude lower than the corresponding kinetic constant of its full-length counterpart. The conformational rearrangements, if any, of enzyme tryptophanes triggered by DNA binding or extension were assayed under pre-steady-state conditions...
September 20, 2017: Protein Journal
https://www.readbyqxmd.com/read/28932213/petri-net-based-model-of-helicobacter-pylori-mediated-disruption-of-tight-junction-proteins-in-stomach-lining-during-gastric-carcinoma
#6
Anam Naz, Ayesha Obaid, Faryal M Awan, Aqsa Ikram, Jamil Ahmad, Amjad Ali
Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28931328/kinetic-and-thermodynamic-parameters-and-partial-characterization-of-the-crude-extract-of-tannase-produced-by-saccharomyces-cerevisiae-ccmb-520
#7
Lúzia Morgana de Melo Lopes, Larissa Hayannyelly Costa Batista, Marcos Juliano Gouveia, Tonny Cley Campos Leite, Marcelo Rodrigues Figueira de Mello, Sandra Aparecida de Assis, Amanda Reges de Sena
Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na(+), Ca(2+), K(+) at 5 × 10(-3) mol/L. The half-life at 30 °C was 3465...
September 20, 2017: Natural Product Research
https://www.readbyqxmd.com/read/28930453/ab-initio-qm-mm-modelling-of-the-rate-limiting-proton-transfer-step-in-the-deamination-of-tryptamine-by-aromatic-amine-dehydrogenase
#8
Kara Elizabeth Ranaghan, William G Morris, Laura Masgrau, Kittusamy Senthilkumar, Linus O Johannissen, Nigel S Scrutton, Jeremy N Harvey, Frederick R Manby, Adrian J Mulholland
Aromatic amine dehydrogenase (AADH) and related enzymes are at the heart of debates on the roles of quantum tunnelling and protein dynamics in catalysis. The reaction of tryptamine in AADH involves significant quantum tunnelling in the rate-limiting proton transfer step, shown e.g. by large H/D primary kinetic isotope effects (KIEs), with unusual temperature dependence. We apply correlated ab initio combined quantum mechanics/molecular mechanics (QM/MM) methods, at levels up to local coupled cluster theory (LCCSD(T)/(aug)-cc-pVTZ), to calculate accurate potential energy surfaces for this reaction, which are necessary for quantitative analysis of tunnelling contributions and reaction dynamics...
September 20, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28929978/a-new-study-of-the-kinetics-of-curd-production-in-the-process-of-cheese-manufacture
#9
Susana Vargas Muñoz, Maykel González Torres, Francisco Quintanilla Guerrero, Rogelio Rodríguez Talavera
We studied the role played by temperature and rennet concentration in the coagulation process for cheese manufacture and the evaluation of their kinetics. We concluded that temperature is the main factor that determines the kinetics. The rennet concentration was unimportant probably due to the fast action of the enzyme chymosin. The Dynamic light scattering technique allowed measuring the aggregate's size and their formation kinetics. The volume fraction of solids was determined from viscosity measurements, showing profiles that are in agreement with the size profiles...
September 20, 2017: Journal of Dairy Research
https://www.readbyqxmd.com/read/28928527/lipase-catalyzed-transesterification-of-ethyl-butyrate-synthesis-in-n-hexane-a-kinetic-study
#10
N Annapurna Devi, G B Radhika, R J Bhargavi
Kinetics of lipase catalyzed transesterification of ethyl caprate and butyric acid was investigated. The objective of this work was to propose a reaction mechanism and develop a rate equation for the synthesis of ethyl butyrate by transesterification using surfactant coated lipase from Candida rugosa. The reaction rate could be described in terms of Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. The values of kinetic parameters computed were Vmax = 2...
August 2017: Journal of Food Science and Technology
https://www.readbyqxmd.com/read/28928426/cyanobacterial-sfp-type-phosphopantetheinyl-transferases-functionalize-carrier-proteins-of-diverse-biosynthetic-pathways
#11
Guang Yang, Yi Zhang, Nicholas K Lee, Monica A Cozad, Sara E Kearney, Hendrik Luesch, Yousong Ding
Cyanobacteria produce structurally and functionally diverse polyketides, nonribosomal peptides and their hybrids. Sfp-type phosphopantetheinyl transferases (PPTases) are essential to the production of these compounds via functionalizing carrier proteins (CPs) of biosynthetic megaenzymes. However, cyanobacterial Sfp-type PPTases remain poorly characterized, posing a significant barrier to the exploitation of cyanobacteria for biotechnological and biomedical applications. Herein, we describe the detailed characterization of multiple cyanobacterial Sfp-type PPTases that were rationally selected...
September 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28928403/dimerization-regulates-both-deaminase-dependent-and-deaminase-independent-hiv-1-restriction-by-apobec3g
#12
Michael Morse, Ran Huo, Yuqing Feng, Ioulia Rouzina, Linda Chelico, Mark C Williams
APOBEC3G (A3G) is a human enzyme that inhibits human immunodeficiency virus type 1 (HIV-1) infectivity, in the absence of the viral infectivity factor Vif, through deoxycytidine deamination and a deamination-independent mechanism. A3G converts from a fast to a slow binding state through oligomerization, which suggests that large A3G oligomers could block HIV-1 reverse transcriptase-mediated DNA synthesis, thereby inhibiting HIV-1 replication. However, it is unclear how the small number of A3G molecules found in the virus could form large oligomers...
September 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28927704/the-catalytic-cycle-of-nitrous-oxide-reductase-the-enzyme-that-catalyzes-the-last-step-of-denitrification
#13
Cíntia Carreira, Sofia R Pauleta, Isabel Moura
The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper "CuZ center"...
September 11, 2017: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/28927325/molecular-imaging-of-hydrolytic-enzymes-using-pet-and-spect
#14
Brian P Rempel, Eric W Price, Christopher P Phenix
Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes...
January 2017: Molecular Imaging
https://www.readbyqxmd.com/read/28925930/in-vitro-glucuronidation-of-wushanicaritin-by-liver-microsomes-intestine-microsomes-and-expressed-human-udp-glucuronosyltransferase-enzymes
#15
Xiaodan Hong, Yuanru Zheng, Zifei Qin, Baojian Wu, Yi Dai, Hao Gao, Zhihong Yao, Frank J Gonzalez, Xinsheng Yao
Wushanicaritin, a natural polyphenol compound, exerts many biological activities. This study aimed to characterize wushanicaritin glucuronidation by pooled human liver microsomes (HLM), human intestine microsomes and individual uridine diphosphate-glucuronosyltransferase (UGT) enzyme. Glucuronidation rates were determined by incubating wushanicaritin with uridine diphosphoglucuronic acid-supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. Reaction phenotyping, the relative activity factor (RAF) and activity correlation analysis were performed to identify the main UGT isoforms...
September 19, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28925864/kinetic-characterization-of-anticarsia-gemmatalis-digestive-serine-proteases-and-the-inhibitory-effect-of-synthetic-peptides
#16
Adriana Patarroyo Vargas, Yaremis Meriño Cabrera, José Cola Zanuncio, Francelina Rocha, Wellington Garcia Campos, Maria Goreti de Almeida Oliveira
The kinetics of Anticarsia gemmatalis Hübner gut serine proteases with tripeptidyl substrates, and the enzymatic inhibition model using synthetic peptides Gor 3, Gor 4, and Gor 5 are described. The enzymes were purified on p-aminobenzamidine agarose affinity column in an FPLC system using electrophoresis with 12.5% polyacrylamide gel. Michaelis-Menten constants and the inhibition model were determined according to the Dixon methodology and Lineweaver-Burk's double reciprocal. The KM values and catalytic constants of peptide substrates show that A...
September 17, 2017: Protein and Peptide Letters
https://www.readbyqxmd.com/read/28925695/nanomolar-inhibitors-of-glycogen-phosphorylase-based-on-%C3%AE-d-glucosaminyl-heterocycles-a-combined-synthetic-enzyme-kinetic-and-protein-crystallography-study
#17
Eva Bokor, Efthimios Kyriakis, Theodora Ga Solovou, Csenge Koppány, Anastassia L Kantsadi, Katalin E Szabó, Andrea Szakács, Georgios A Stravodimos, Tibor Docsa, Vassiliki T Skamnaki, Spyros E Zographos, Pál Gergely, Demetres D Leonidas, László Somsák
Aryl substituted 1-(β-D-glucosaminyl)-1,2,3-triazoles as well as C-β-D-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-β-D-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-β-D-glucosaminyl derivatives had potent activity and the best inhibitor was the 2-(β-D-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki value of 143 nM against human liver GPa. An X-ray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding, and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues...
September 19, 2017: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/28924834/diversity-of-family-gh46-chitosanases-in-kitasatospora-setae-km-6054
#18
Mina Zitouni, Pascal Viens, Mariana G Ghinet, Ryszard Brzezinski
The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid...
September 18, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28924503/biochemical-and-structural-characterization-of-a-novel-arginine-kinase-from-the-spider-polybetes-pythagoricus
#19
Aldana Laino, Alonso A Lopez-Zavala, Karina D Garcia-Orozco, Jesus S Carrasco-Miranda, Marianela Santana, Vivian Stojanoff, Rogerio R Sotelo-Mundo, Carlos Fernando Garcia
Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure...
2017: PeerJ
https://www.readbyqxmd.com/read/28924089/synthesis-of-chirally-pure-enantiomers-by-lipase
#20
Kamal Kumar Bhardwaj, Reena Gupta
Pure enantiomers can be produced by chemical and enzymatic methods. Enzymatic methods to produce pure enantiomers are more advantageous. High enantiomeric excess is the advantage of enzymatic method, resulting from the inherent selectivity of the enzyme. Lipase (EC 3.1.1.3) enzymes are widely used for the synthesis of chirally pure enantiomers because of their stereoselectivities which can satisfy the growing demand for obtaining optically active compounds. Various methods are used for the synthesis of chirally pure enantiomers, however kinetic resolution and asymmetric synthesis are more common...
September 15, 2017: Journal of Oleo Science
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