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Enzyme kinetics

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https://www.readbyqxmd.com/read/28214753/structural-and-functional-insights-into-corrinoid-iron-sulfur-protein-from-human-pathogen-clostridium-difficile
#1
Yaozhu Wei, Xiaofei Zhu, Sixue Zhang, Xiangshi Tan
The human pathogen Clostridium difficile infection (CDI) is one of the most important healthcare-associated infections. The Wood-Ljungdahl pathway, which is responsible for Acetyl-CoA biosynthesis, is essential for the survival of the pathogen and is absent in humans. The key proteins and enzymes involved in the pathway are attractive targets for the treatment of CDI. Corrinoid iron-sulfur protein (CoFeSP) is a key protein and acts as a methyl transformer in the Wood-Ljungdahl pathway. In this study, CoFeSP from Clostridium difficile (CoFeSPCd) was cloned, expressed in E...
February 13, 2017: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/28213218/dynamic-flow-through-approach-to-evaluate-readily-bioaccessible-antioxidants-in-solid-food-samples
#2
Miguel A Maia, Tânia R P Soares, Ana I P Mota, María Rosende, Luís M Magalhães, Manuel Miró, Marcela A Segundo
Release of bioactive compounds from food matrices is regarded as the first step towards their human bioavailability. The objective of this work was the implementation of an affordable and robust flow-through device for expedient dynamic leaching experiments aiming at the assessment of readily bioaccessible antioxidant compounds in solid food commodities. A simple configuration is proposed using commercially available devices containing regenerated cellulose filters placed in polypropylene holders to entrap the solid sample, featuring a disposable, single use extraction chamber...
May 1, 2017: Talanta
https://www.readbyqxmd.com/read/28212377/optimality-principles-reveal-a-complex-interplay-of-intermediate-toxicity-and-kinetic-efficiency-in-the-regulation-of-prokaryotic-metabolism
#3
Jan Ewald, Martin Bartl, Thomas Dandekar, Christoph Kaleta
A precise and rapid adjustment of fluxes through metabolic pathways is crucial for organisms to prevail in changing environmental conditions. Based on this reasoning, many guiding principles that govern the evolution of metabolic networks and their regulation have been uncovered. To this end, methods from dynamic optimization are ideally suited since they allow to uncover optimality principles behind the regulation of metabolic networks. We used dynamic optimization to investigate the influence of toxic intermediates in connection with the efficiency of enzymes on the regulation of a linear metabolic pathway...
February 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28212001/rapid-method-for-the-quantification-of-reduced-and-oxidized-glutathione-in-human-plasma-and-saliva
#4
Kamonwad Ngamchuea, Christopher Batchelor-McAuley, Richard G Compton
A new method is developed to determine the concentrations of reduced (GSH) and oxidized glutathione (GSSG), and it enables the calculation of the GSH:GSSG ratios in human plasma and saliva samples. The assay is based on the masking of GSH in a GSH and GSSG mixture via a 1,4-addition reaction with p-benzoquinone (BQ), followed by enzymatic kinetic measurement. The enzyme, glutathione reductase, is highly specific to glutathione. Excess BQ can thus be easily removed by the addition of non-GSH thiols. The assay takes less than 2 min, is suitable for a short-time-scale study, and minimizes the in vitro underestimation of the GSH:GSSG ratio arising from the degradation of GSH and formation of GSSG...
February 17, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28211541/antibody-controlled-actuation-of-dna-based-molecular-circuits
#5
Wouter Engelen, Lenny H H Meijer, Bram Somers, Tom F A de Greef, Maarten Merkx
DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically...
February 17, 2017: Nature Communications
https://www.readbyqxmd.com/read/28208301/biomolecular-interaction-assays-identified-dual-inhibitors-of-glutaminase-and-glutamate-dehydrogenase-that-disrupt-mitochondrial-function-and-prevent-growth-of-cancer-cells
#6
Min Zhu, Jinzhang Fang, Jingjing Zhang, Zheng Zhang, Jianhui Xie, Yan Yu, Jennifer Jin Ruan, Zhao Chen, Wei Hou, Gensheng Yang, Weike Su, Benfang Helen Ruan
Glutaminase (KGA/isoenzyme GAC) is an emerging and important drug target for cancer. Traditional methods for assaying glutaminase activity are coupled with several other enzymes. Such coupled assays do not permit the direct and stringent characterization of specific glutaminase inhibitors. Ebselen was identified as a potent 9 nM KGA inhibitor in the KGA/glutamate oxidase (GO)/horse radish peroxidase (HRP) coupled assay but showed very weak activity in inhibiting the growth of glutamine-dependent cancer cells...
February 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28207833/structure-kinetic-characterization-and-subcellular-localization-of-the-two-ribulose-5-phosphate-epimerase-isoenzymes-from-trypanosoma-cruzi
#7
Soledad Natalia Gonzalez, Wanda Mariela Valsecchi, Dante Maugeri, José María Delfino, Juan José Cazzulo
The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms...
2017: PloS One
https://www.readbyqxmd.com/read/28207324/is-serum-angiotensin-converting-enzyme-level-useful-for-determining-necroinflammatory-activity-in-chronic-hepatitis-b-infection
#8
Ahmet Tay, Fatih Albayrak, Sevilay Ozmen, Ayse Albayrak, Kemalettin Ozden
AIM: The purpose of this study was to investigate the relationship between the findings from liver biopsy and the serum angiotensin-converting enzyme (ACE) level to determine whether ACE might serve as a potential noninvasive sign of necroinflammatory activity in patients with Chronic Hepatitis B (CHB) infection. METHODS: A total of 54 CHB patients referred for liver biopsy were enrolled in the study. Serum ACE levels were determined photometrically with a kinetic test...
February 2017: Genetic Testing and Molecular Biomarkers
https://www.readbyqxmd.com/read/28202545/the-basis-for-acyl-specificity-in-the-tafazzin-reaction
#9
Michael Schlame, Yang Xu, Mindong Ren
Tafazzin is a mitochondrial enzyme that transfers fatty acids from phospholipids to lysophospholipids. Mutations in tafazzin cause abnormal molecular species of cardiolipin and the clinical phenotype of Barth syndrome. However, the mechanism by which tafazzin creates acyl specificity has been controversial. We have shown that the lipid phase state can produce acyl specificity in the tafazzin reaction but others have reported that tafazzin itself carries enzymatic specificity. To resolve this issue, we replicated and expanded the controversial experiments, i...
February 15, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28202305/enzymatic-synthesis-of-2-o-methylribonucleosides-with-a-nucleoside-hydrolase-family-enzyme-from-lactobacillus-buchneri-lbk78
#10
Yuuki Mitsukawa, Makoto Hibi, Narihiro Matsutani, Nobuyuki Horinouchi, Satomi Takahashi, Jun Ogawa
2'-O-Methylribonucleosides (2'-OMe-NRs) are promising raw materials for the production of nucleic acid drugs. We previously reported that LbNH, a nucleoside hydrolase from Lactobacillus buchneri LBK78 (NITE P-01581), was the first enzyme found to act on 2'-OMe-NRs. In the present study, we determined that LbNH also has the transribosylation activity between 2'-OMe-NRs and nucleobases, in addition to the hydrolyzing activity towards 2'-OMe-NRs. When 2'-O-methyluridine (2'-OMe-UR) and adenine were reacted with LbNH, 2'-O-methyladenosine (2'-OMe-AR) was produced...
February 12, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28201966/synthesis-and-biological-evaluation-of-mutual-prodrugs-of-carboxylic-group-containing-some-non-steroidal-anti-inflammatory-drugs-and-propyphenazone
#11
Sucheta Ohlan, Meenu Paliwal, Ruchita Ruchita, Shilpa Jain, Monika Monika, Himanshu Himanshu
Non-steroidal anti-inflammatory drugs (NSAIDs); ketorolac (KA), niflumic acid (NA), tolfenamic acid (TA) were conjugated with propyphenazone derivative 3-Hydroxymethyl propyphenazone (HMP) by direct coupling and by using spacer technique with the objective of obtaining gastro-sparing NSAIDs prodrugs devoid of ulcerogenic side effects. Mutual prodrugs were synthesized and their structures were confirmed and characterized using IR, 1H NMR, 13C NMR, mass spectroscopy and their purity was established by elemental analysis...
February 13, 2017: Current Drug Delivery
https://www.readbyqxmd.com/read/28199119/engineering-a-catalytically-efficient-recombinant-protein-ligase
#12
Renliang Yang, Yee Hwa Wong, Giang K T Nguyen, James P Tam, Julien Lescar, Bin Wu
Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its pre-activation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity...
February 15, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28196894/extending-enzyme-molecular-recognition-with-an-expanded-amino-acid-alphabet
#13
Claire L Windle, Katie J Simmons, James R Ault, Chi H Trinh, Adam Nelson, Arwen R Pearson, Alan Berry
Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates...
February 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28195477/enzymatic-n-and-c-protection-in-cyanobactin-ripp-natural-products
#14
Debosmita Sardar, Yue Hao, Zhenjian Lin, Maho Morita, Satish K Nair, Eric W Schmidt
Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products...
February 15, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28195196/arabidopsis-thaliana-dehydroascorbate-reductase-2-conformational-flexibility-during-catalysis
#15
Nandita Bodra, David Young, Leonardo Astolfi Rosado, Anna Pallo, Khadija Wahni, Frank De Proft, Jingjing Huang, Frank Van Breusegem, Joris Messens
Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity...
February 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28194412/effect-of-r119g-mutation-on-human-p5cr1-dynamic-property-and-enzymatic-activity
#16
Linhua Li, Yujia Ye, Peng Sang, Yirui Yin, Wei Hu, Jing Wang, Chao Zhang, Deyun Li, Wen Wan, Rui Li, Longjun Li, Linling Ma, Yuehui Xie, Zhaohui Meng
Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28193496/development-of-a-colorimetric-assay-for-glycosynthases
#17
Marc R Hayes, Kevin Bochinsky, Lisa S Seibt, Lothar Elling, Jörg Pietruszka
The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration...
February 10, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28193420/reaction-kinetics-and-galactooligosaccharide-product-profiles-of-the-%C3%AE-galactosidases-from-bacillus-circulans-kluyveromyces-lactis-and-aspergillus-oryzae
#18
Huifang Yin, Jelle B Bultema, Lubbert Dijkhuizen, Sander S van Leeuwen
β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19...
June 15, 2017: Food Chemistry
https://www.readbyqxmd.com/read/28193403/thermal-inactivation-kinetics-of-%C3%AE-galactosidase-during-bread-baking
#19
Lu Zhang, Xiao Dong Chen, Remko M Boom, Maarten A I Schutyser
In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R(2)=0...
June 15, 2017: Food Chemistry
https://www.readbyqxmd.com/read/28193333/heterologous-expression-of-%C3%AE-glutamyl-transpeptidase-from-bacillus-atrophaeus-gs-16-and-its-application-in-the-synthesis-of-%C3%AE-d-glutamyl-l-tryptophan-a-known-immunomodulatory-peptide
#20
Meenu Saini, Shruti Bindal, Rani Gupta
Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5...
April 2017: Enzyme and Microbial Technology
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