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Enzyme kinetics

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https://www.readbyqxmd.com/read/28103425/efficient-synthesis-of-%C3%AE-galactosyl-oligosaccharides-using-a-mutant-bacteroides-thetaiotaomicron-retaining-%C3%AE-galactosidase-btgh97b
#1
Masayuki Okuyama, Kana Matsunaga, Ken-Ichi Watanabe, Keitaro Yamashita, Takayoshi Tagami, Asako Kikuchi, Min Ma, Patcharapa Klahan, Haruhide Mori, Min Yao, Atsuo Kimura
The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α-glycosidases can give poor yields of desired products because they require generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors...
January 19, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28102965/isotope-probing-of-the-udp-apiose-udp-xylose-synthase-reaction-evidence-of-a-mechanism-via-a-coupled-oxidation-and-aldol-cleavage
#2
Thomas Eixelsberger, Doroteja Horvat, Alexander Gutmann, Hansjörg Weber, Bernd Nidetzky
The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively (2) H-labeled and deoxygenated substrates. The analogue UDP-2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the plausible initiating step of pyranoside-to-furanoside conversion, did not give the corresponding Api product...
January 19, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28102315/methyl-cytosine-driven-structural-changes-enhance-adduction-kinetics-of-an-exon-7-fragment-of-the-p53-gene
#3
Spundana Malla, Karteek Kadimisetty, You-Jun Fu, Dharamainder Choudhary, John B Schenkman, James F Rusling
Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites...
January 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28100776/crystallographic-snapshots-of-class-a-%C3%AE-lactamase-catalysis-reveal-structural-changes-that-facilitate-%C3%AE-lactam-hydrolysis
#4
Xuehua Pan, Yunjiao He, Jinping Lei, Xuhui Huang, Yanxiang Zhao
β-lactamases confer resistance to β-lactam-based antibiotics. There is great interest in understanding their mechanisms to enable the development of β-lactamase-specific inhibitors. The mechanism of Class A β-lactamases has been studied extensively, revealing Lys73 and Glu166 as general bases that assist the catalytic residue Ser70. However, the specific roles of these two residues within the catalytic cycle remains not fully understood. To help resolve this question, we first identified a Glu166His mutant that is functional but kinetically slowed...
January 18, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28100651/stabilization-of-bacillus-circulans-xylanase-by-combinatorial-insertional-fusion-to-a-thermophilic-host-protein
#5
Vandan Shah, Brennal Pierre, Tamari Kirtadze, Seung Shin, Jin Ryoun Kim
High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX)...
January 17, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28098999/dna-deformation-coupled-recognition-of-8-oxoguanine-conformational-kinetic-gating-in-human-dna-glycosylase
#6
Haoquan Li, Anton V Endutkin, Christina Bergonzo, Fu Lin, Arthur P Grollman, Dmitry O Zharkov, Carlos Simmerling
8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 and H7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine-DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition...
January 18, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28098963/scaffolding-of-enzymes-on-virus-nanoarrays-effects-of-confinement-and-virus-organization-on-biocatalysis
#7
Anisha N Patel, Agnès Anne, Arnaud Chovin, Christophe Demaille, Eric Grelet, Thierry Michon, Cécilia Taofifenua
Organizing active enzyme molecules on nanometer-sized scaffolds is a promising strategy for designing highly efficient supported catalytic systems for biosynthetic and sensing applications. This is achieved by designing model nanoscale enzymatic platforms followed by thorough analysis of the catalytic activity. Herein, the virus fd bacteriophage is considered as an enzyme nanocarrier to study the scaffolding effects on enzymatic activity. Nanoarrays of randomly oriented, or directionally patterned, fd bacteriophage virus are functionalized with the enzyme glucose oxidase (GOx), using an immunological assembly strategy, directly on a gold electrode support...
January 18, 2017: Small
https://www.readbyqxmd.com/read/28098838/role-of-udp-glucuronosyltransferase-1a1-in-the-metabolism-and-pharmacokinetics-of-silymarin-flavonolignans-in-patients-with-hcv-and-nafld
#8
Ying Xie, Sonia R Miranda, Janelle M Hoskins, Roy L Hawke
Silymarin is the most commonly used herbal medicine by patients with chronic liver disease. Silymarin flavonolignans undergo rapid first-pass metabolism primarily by glucuronidation. The aims of this investigation were: (1) to determine the association of UGT1A1*28 polymorphism with the area under the plasma concentration-time curves (AUCs) for silybin A (SA) and silybin B (SB); (2) to evaluate the effect of UGT1A1*28 polymorphism on the profile of flavonolignan glucuronide conjugates found in the plasma; and (3) to investigate the role of UGT1A1 enzyme kinetics on the pharmacokinetics of SA and SB...
January 15, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28098357/partial-purification-characterization-and-thermal-inactivation-kinetics-of-peroxidase-and-polyphenol-oxidase-isolated-from-kalipatti-sapota-manilkara-zapota
#9
Vishwasrao Chandrahas, Chakraborty Snehasis, Ananthanarayan Laxmi
BACKGROUND: The extraction, purification, and characterization of peroxidase (POD) and polyphenol oxidase (PPO) were studied for Kalipatti sapota fruit. The crude enzyme extract was partially purified by ammonium sulfate precipitation followed by BioGel P100 size exclusion and Unosphere Q anion-exchange chromatography. RESULTS: Molecular weights of 20 kDa (POD) and 24 kDa (PPO) were indicated by SDS-PAGE. A single band was observed on SDS-PAGE with a fold purity of 10...
January 18, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28098337/characterisation-of-the-enzymatic-properties-of-mpapr1-an-aspartic-protease-secreted-by-the-wine-yeast-metschnikowia-pulcherrima
#10
Louwrens Wiid Theron, Marina Bely, Benoit Divol
BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3...
January 18, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28098230/molecular-mechanism-of-the-allosteric-regulation-of-the-%C3%AE-%C3%AE-heterodimer-of-human-nad-dependent-isocitrate-dehydrogenase
#11
Tengfei Ma, Yingjie Peng, Wei Huang, Jianping Ding
Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Previously, we have demonstrated biochemically that the α2βγ heterotetramer and αγ heterodimer can be allosterically activated by citrate (CIT) and ADP. In this work, we report the crystal structures of the αγ heterodimer with the γ subunit bound without or with different activators...
January 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28098152/human-farnesyl-pyrophosphate-synthase-is-allosterically-inhibited-by-its-own-product
#12
Jaeok Park, Michal Zielinski, Alexandr Magder, Youla S Tsantrizos, Albert M Berghuis
Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme's active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The Kd for this binding is 5-6 μM, within a catalytically relevant range...
January 18, 2017: Nature Communications
https://www.readbyqxmd.com/read/28097915/the-relationship-between-angiotensin-converting-enzyme-ace-insertion-i-deletion-d-polymorphism-serum-ace-activity-and-bone-mineral-density-bmd-in-older-chinese
#13
Ya-Feng Zhang, Hong Wang, Qiong Cheng, Ling Qin, Nelson Ls Tang, Ping-Chong Leung, Timothy Cy Kwok
OBJECTIVE: In this study, we set out to investigate the relationship between angiotensin-converting enzyme ( ACE) I/D polymorphism, serum ACE activity and bone mineral density (BMD) in older Chinese. METHODS: A standardized, structured, face-to-face interview was performed to collect demographic information. BMD was measured using dual-energy X-ray absorptiometry (DXA). I/D genotypes of ACE were determined by polymerase chain reaction (PCR) amplification. Serum ACE activity was determined photometrically by a commercially available kinetic kit...
January 2017: Journal of the Renin-angiotensin-aldosterone System: JRAAS
https://www.readbyqxmd.com/read/28097911/design-synthesis-and-docking-study-of-novel-coumarin-ligands-as-potential-selective-acetylcholinesterase-inhibitors
#14
Fatih Sonmez, Belma Zengin Kurt, Isil Gazioglu, Livia Basile, Aydan Dag, Valentina Cappello, Tiziana Ginex, Mustafa Kucukislamoglu, Salvatore Guccione
New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death...
December 2017: Journal of Enzyme Inhibition and Medicinal Chemistry
https://www.readbyqxmd.com/read/28097167/expression-purification-and-kinetic-characterization-of-recombinant-benzoate-dioxygenase-from-rhodococcus-ruber-ukmp-5m
#15
Arezoo Tavakoli, Ainon Hamzah, Amir Rabu
In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3)...
September 2016: Molecular Biology Research Communications
https://www.readbyqxmd.com/read/28096941/transport-of-biomolecules-to-binding-partners-displayed-on-the-surface-of-microbeads-arrayed-in-traps-in-a-microfluidic-cell
#16
Xiaoxiao Chen, Thomas F Leary, Charles Maldarelli
Arrays of probe molecules integrated into a microfluidic cell are utilized as analytical tools to screen the binding interactions of the displayed probes against a target molecule. These assay platforms are useful in enzyme or antibody discovery, clinical diagnostics, and biosensing, as their ultraminiaturized design allows for high sensitivity and reduced consumption of reagents and target. We study here a platform in which the probes are first grafted to microbeads which are then arrayed in the microfluidic cell by capture in a trapping course...
January 2017: Biomicrofluidics
https://www.readbyqxmd.com/read/28095673/a-comparative-study-of-hepatotoxicity-of-pyrrolizidine-alkaloids-retrorsine-and-monocrotaline
#17
Xiaojing Yang, Weiwei Li, Ying Sun, Xiucai Guo, Wenlin Huang, Ying Peng, Jiang Zheng
Many pyrrolizidine alkaloids (PAs) can cause liver injury in animals and humans. Different hepatotoxic PAs can produce similar hepatotoxic effects, but the degree of their toxicities may vary widely. Retrorsine (RTS) and monocrotaline (MCT) share the same core structure (retronecine) and similar metabolic activation pathway. RTS and MCT both produced liver injury but the former was more hepatotoxic than the latter. Enzyme kinetic study demonstrated that the value of Vmax/Km for RTS was 5.5-fold larger than that of MCT...
January 17, 2017: Chemical Research in Toxicology
https://www.readbyqxmd.com/read/28095341/effect-of-the-r119g-mutation-on-human-p5cr-structure-and-its-interactions-with-nad-insights-derived-from-molecular-dynamics-simulation-and-free-energy-analysis
#18
Peng Sang, Yue-Hui Xie, Lin-Hua Li, Yu-Jia Ye, Wei Hu, Jing Wang, Wen Wan, Rui Li, Long-Jun Li, Lin-Ling Ma, Zhi Li, Shu-Qun Liu, Zhao-Hui Meng
Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex...
January 5, 2017: Computational Biology and Chemistry
https://www.readbyqxmd.com/read/28093699/anti-alzheimer-s-disease-activity-of-compounds-from-the-root-bark-of-morus-alba-l
#19
Eun Bi Kuk, A Ra Jo, Seo In Oh, Hee Sook Sohn, Su Hui Seong, Anupom Roy, Jae Sue Choi, Hyun Ah Jung
The inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays important roles in prevention and treatment of Alzheimer's disease (AD). Among the individual parts of Morus alba L. including root bark, branches, leaves, and fruits, the root bark showed the most potent enzyme inhibitory activities. Therefore, the aim of this study was to evaluate the anti-AD activity of the M. alba root bark and its isolate compounds, including mulberrofuran G (1), albanol B (2), and kuwanon G (3) via inhibition of AChE, BChE, and BACE1...
January 16, 2017: Archives of Pharmacal Research
https://www.readbyqxmd.com/read/28093470/active-site-maturation-and-activity-of-the-copper-radical-oxidase-glxa-is-governed-by-a-tryptophan-residue
#20
Amanda K Chaplin, Dimitri A Svistunenko, Michael Hough, Michael T Wilson, Erik Vijgenboom, Jonathan Ar Worrall
GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best-characterized. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic site in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine...
January 16, 2017: Biochemical Journal
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