DOCSLIB.ORG

On the Active Site Thiol of Y-Glutamylcysteine Synthetase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
On the Active Site Thiol of Y-Glutamylcysteine Synthetase Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2464-2468, April 1988 Biochemistry On the active site thiol of y-glutamylcysteine synthetase: Relationships to catalysis, inhibition, and regulation (glutathione/cystamine/Escherichia coli/kidney/enzyme inactivation) CHIN-SHIou HUANG, WILLIAM R. MOORE, AND ALTON MEISTER Cornell University Medical College, Department of Biochemistry, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 4, 1987 ABSTRACT y-Glutamylcysteine synthetase (glutamate- dithiothreitol, suggesting that cystamine forms a mixed cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia disulfide between cysteamine and an enzyme thiol (15). coli strain enriched in the gene for this enzyme by recombinant Inactivation of the enzyme by the L- and D-isomers of DNA techniques. The purified enzyme has a specific activity of 3-amino-1-chloro-2-pentanone, as well as that by cystamine, 1860 units/mg and a molecular weight of 56,000. Comparison is prevented by L-glutamate (14). Treatment of the enzyme of the E. coli enzyme with the well-characterized rat kidney with cystamine prevents its interaction with the sulfoxi- enzyme showed that these enzymes have similar catalytic prop- mines. Titration of the enzyme with 5,5'-dithiobis(2- erties (apparent Km values, substrate specificities, turnover nitrobenzoate) reveals that the enzyme has a single exposed numbers). Both enzymes are feedback-inhibited by glutathione thiol that reacts with this reagent without affecting activity but not by y-glutamyl-a-aminobutyrylglycine; the data indicate (16). 5,5'-Dithiobis(2-nitrobenzoate) does not interact with that glutathione binds not only at the glutamate binding site but the thiol that reacts with cystamine. Evidence has also been also at a second site on the enzyme that interacts with the thiol obtained that y-methylene-D-glutamate, which inactivates moiety of glutathione but not with a methyl group. Both the enzyme, binds in the glutamate binding site of the active enzymes are inactivated by buthionine sulfoximine in the pres- center and forms a covalent linkage with the active site thiol ence of ATP, suggesting a common y-glutamyl phosphate through a Michael-type addition reaction (17). The enzyme is intermediate. However, unlike the rat kidney enzyme that has also inactivated by S-sulfocysteine and S-sulfohomocys- an active center thiol, the bacterial enzyme is insensitive to teine; these inactivators are tightly but noncovalently bound cystamine, r-methylene glutamate, and S-sulfo amino acids, and their interaction with the enzyme is postulated to indicating that it does not have an active site thiol. Thus, the rat involve effects of the active site thiol (18). These and earlier kidney and E. coli enzymes share several catalytic features but (14) observations have led to the suggestion that the active differ in active site structure. If the active site thiol of the rat center thiol may play a role in the mechanism of action of is in this enzyme. kidney enzyme involved catalysis, which seems likely, there In contrast to the results summarized above obtained in would appear to be differences in the mechanisms of action of studies on rat kidney y-glutamylcysteine synthetase, the the two y-glutamylcysteine synthetases. findings on the y-glutamylcysteine synthetase ofEscherichia coli, reported here, indicate that the bacterial enzyme has The first step in the synthesis of glutathione is catalyzed by properties that do not reflect the presence ofan active center y-glutamylcysteine synthetase (glutamate-cysteine ligase; thiol. Nevertheless, the bacterial enzyme exhibits a number EC 6.3.2.2) [Reaction 1 (ref. 1, pp. 671-697)]. This reaction, of characteristics that closely resemble those of the mam- usually the rate-limiting step in glutathione synthesis, is malian enzyme, including, for example, high turnover num- feedback-inhibited by glutathione (2). y-Glutamylcysteine ber, feedback inhibition by glutathione, and inactivation by synthetase, like glutamine synthetase (ref. 1, pp 699-754, incubation with buthionine sulfoximine in the presence of and refs. 3 and 4), is inactivated by methionine sulfoximine ATP. A preliminary account of this work has been presented in the presence of ATP (5). Methionine sulfoximine and (*; ref. 19). certain other sulfoximines, such as buthionine sulfoximine [which inhibits y-glutamylcysteine synthetase but not gluta- EXPERIMENTAL PROCEDURES mine synthetase (6-9)], are phosphorylated by ATP on the enzymes; the phosphorylated sulfoximines bind tightly, but Materials. Agarose-hexane-ATP (Al6; type 2), CM-Seph- noncovalently, to the enzymes, thus producing inhibition. adex C-50, Sephadex G-100, and Sephadex G-150 were purchased from Pharmacia. DE-52 was obtained from What- L-Glutamate + ATP + L-cysteine man. L-Buthionine-(SR)-sulfoximine (6-8), S-sulfocysteine L-y-glutamyl-L-cysteine + ADP + Pi [1] (18), S-sulfohomocysteine (18), and y-methylene glutamate (17) were synthesized as described. Other compounds were The presence of a thiol at the active site of y-glutamyl- obtained from Sigma. cysteine synthetase was suggested by the finding (10) that Microorganisms. Previously a strain of E. coli enriched by highly purified 'y-glutamylcysteine synthetase from rat kid- recombinant DNA techniques in its content of the two ney is inactivated by low concentrations of L-2-amino-4-oxo- synthetases required for glutathione synthesis was described 5-chloropentanoate (11), that such inactivation is associated (20, 21). The genes for the synthetases were isolated and with covalent binding of the inactivator, and that glutamate inserted into a vector that was used to transform the original protects against inactivation. Subsequently it was found that strain. The transformed strain, in an immobilized form, has the enzyme is strongly inactivated by cystamine (12-14) and been used for the synthesis of isotopically labeled gluta- that inactivation by cystamine is reversed by treatment with thione (22) and for the synthesis of glutathione analogs (23). In the present work a bacterial strain enriched in its content The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *Huang, C.-S., Moore, W. R. & Meister, A., American Society of in accordance with 18 U.S.C. §1734 Alely to indicate this fact. Biological Chemists Meeting, June 6-10, 1987, Philadelphia, PA. 2464 Downloaded by guest on October 4, 2021 OXYGEN COST AND EFFICIENCY IN HEALTH AND EMPHYSEMA 497 z 0 Z 0 - 7s I/ . / II100 200. O 0 0E NO1/ d)/ P1 80 10V CLJ 0 2 150 x 4p,, x > -a Zn 60 - C) u0 -~ 100 -w°X 1. r0 40 IZ I z I . ol 0~. :-0c LA 20 0 10 20 30 4I0 0 6 3 'D VENTILATION A z (L/min. B.T.P.S.) 0 2.0 4.0 6.0 8.0 FIG. 3. THE CHANGE IN OXYGEN CONSUMPTION As- ADDED MECHANICAL WORK SOCIATED WITH CHANGE IN MINUTE VENTILATION IN 22 (kgm.m./min.) PATIENTS WITH EMPHYSEMA FIG. 4. THE EXTRA OXYGEN CONSUMPTION REQUIRED Also shown are isopleths representing the oxygen TO HANDLE ADDED RESPIRATORY WORK LOADS IN NINE cost of per liter of ventilation. breathing NORMAL AND SEVEN EMPHYSEMATOUS SUBJECTS Also shown are isopleths representing the efficiency of tion of the respiratory muscles per liter of ventila- the respiratory muscles for handling added work loads. tion was considerably higher in the patients with 0 represents normal subjects; X represents emphysema- emphysema than in the normal subjects. The tous subjects. cost ranged between 0.45 to 1.87 ml. per L. of ventilation (mean 1.16) in the normal subjects, was high for even slight increases in ventilation while it ranged between 1.68 and 18.50 ml. per L. and rose still further when ventilation was in- of ventilation (mean 5.96) in the emphysematous creased. subjects. The extra oxygen consumption associated with In Figures 2 and 3 the increments in oxygen added work loads in nine normal subjects and consumption associated with changes in ventilation seven patients with emphysema is shown in Fig- in the normal subjects and patients with emphy- ure 4. It can be seen that the extra oxygen sema are shown. It can be seen that in the normal consumption per unit added work load was greater subjects the oxygen cost of an increase in ventila- in the patients with emphysema than in the tion of 30 L. per minute above the resting ventila- normals. tion was less than 2 ml. per L. On the other hand, In Table III the calculated efficiencies of the in the subjects with emphysema the oxygen cost respiratory muscles of the nine normal and seven TABLE III Total mechanical work and efficiency of the respiratory muscles in normal and emphysematous subjects Normal Emphysematous Total mechanical work Total mechanical work Subj. Efficiency at rest Subj. Efficiency at rest % Kg. M./L. Kg. M./min. % Kg. M./L. Kg. M./min. 1 10.80 0.173 1.52 17 2.74 0.141 1.39 2 7.45 0.172 1.38 18 1.45 0.203 1.77 3 7.60 0.123 0.82 19 2.30 0.159 1.39 4 8.20 0.280 2.69 20 2.04 0.120 0.83 5 11.10 0.326 3.00 21 1.24 0.188 1.20 6 8.04 0.204 2.74 22 1.47 0.284 2.68 7 7.20 0.203 1.83 23 1.60 0.123 1.22 8 7.43 0.215 1.89 9 9.35 0.349 2.27 Mean 8.58 0.227 2.02 1.83 0.174 1.49 Downloaded by guest on October 4, 2021 2466 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1.0, and 0.3 mM. The relative rates of reaction found with Table 2.
Load more

Recommended publications
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [Show full text]
  • Methionine Sulfoximine: a Novel Anti Inflammatory Agent
    Wayne State University Wayne State University Dissertations January 2018 Methionine Sulfoximine: A Novel Anti Inflammatory Agent Tyler Peters Wayne State University, [email protected] Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Biochemistry Commons Recommended Citation Peters, Tyler, "Methionine Sulfoximine: A Novel Anti Inflammatory Agent" (2018). Wayne State University Dissertations. 2124. https://digitalcommons.wayne.edu/oa_dissertations/2124 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. METHIONINE SULFOXIMINE: A NOVEL ANTI-INFLAMMATORY AGENT by TYLER J. PETERS DISSERTATION Submitted to the Graduate School of Wayne State University – School of Medicine Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOHPY 2018 MAJOR: BIOCHEMISTRY & MOL. BIOLOGY Approved By: __________________________________________ Advisor Date DEDICATION This work is dedicated to my family. I wouldn’t have made it this far without your unconditional love and support. ii ACKNOWLEDGEMENTS Thank you Dr. Brusilow, I consider myself very fortunate for having the privilege of working in the laboratory of Dr. William S.A. Brusilow these past few years. Under his mentorship, my scientific autonomy was always respected, and my opinions were always valued with consideration. I am thankful for his guidance and support as an advisor; I truly admire his patience and envy his calm demeanor. He exemplifies scientific integrity, and his dedication to develop MSO has inspired me. I had never experienced consistent failure in any aspect of life before encountering scientific research; at times I felt that Dr.
    [Show full text]
  • Crystallography Captures Catalytic Steps in Human Methionine Adenosyltransferase Enzymes
    Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes Ben Murraya,b, Svetlana V. Antonyuka, Alberto Marinab, Shelly C. Luc, Jose M. Matod, S. Samar Hasnaina,1, and Adriana L. Rojasb,1 aMolecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, England; bStructural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain; cDivision of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and dCIC bioGUNE, CIBERehd, Parque Tecnologico Bizkaia, 801A-1.48160 Derio, Spain Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved January 8, 2016 (received for review June 4, 2015) The principal methyl donor of the cell, S-adenosylmethionine catalytic mechanism (13, 14) in which the reaction is initiated (SAMe), is produced by the highly conserved family of methionine through a nucleophilic attack by the sulfur atom of methionine adenosyltranferases (MATs) via an ATP-driven process. These en- on the C5′ atom of ATP, which produces the intermediate tri- zymes play an important role in the preservation of life, and their polyphosphate (PPPi). Hydrolysis of the PPPi into pyrophos- dysregulation has been tightly linked to liver and colon cancers. We phate (PPi) and orthophosphate (Pi) then occurs (Fig. S1). present crystal structures of human MATα2 containing various bound A common feature of MAT enzymes is a gating loop that α – ligands, providing a “structural movie” of the catalytic steps. High- to flanks the active site (in human MAT 2 residues 113 131), atomic-resolution structures reveal the structural elements of the which has been postulated to act in a dynamic way to allow ac- enzyme involved in utilization of the substrates methionine and cess to the active site.
    [Show full text]
  • Review Article Cystathionine -Synthase in Physiology and Cancer
    Hindawi BioMed Research International Volume 2018, Article ID 3205125, 11 pages https://doi.org/10.1155/2018/3205125 Review Article Cystathionine �-Synthase in Physiology and Cancer Haoran Zhu,1,2 Shaun Blake,1,2 Keefe T. Chan,1 Richard B. Pearson ,1,2,3,4 and Jian Kang 1 1 Division of Research, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria 3000, Australia 2Sir Peter MacCallum Department of Oncology, Australia 3Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia 4Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia Correspondence should be addressed to Richard B. Pearson; [email protected] Received 23 March 2018; Accepted 29 May 2018; Published 28 June 2018 Academic Editor: Maria L. Tornesello Copyright © 2018 Haoran Zhu et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cystathionine �-synthase (CBS) regulates homocysteine metabolism and contributes to hydrogen sulfde (H2S) biosynthesis through which it plays multifunctional roles in the regulation of cellular energetics, redox status, DNA methylation, and protein modifcation. Inactivating mutations in CBS contribute to the pathogenesis of the autosomal recessive disease CBS-defcient homocystinuria. Recent studies demonstrating that CBS promotes colon and ovarian cancer growth in preclinical models highlight a newly identifed oncogenic role for CBS. On the contrary, tumor-suppressive efects of CBS have been reported in other cancer types, suggesting context-dependent roles of CBS in tumor growth and progression. Here, we review the physiological functions of CBS, summarize the complexities regarding CBS research in oncology, and discuss the potential of CBS and its key metabolites, including homocysteine and H2S, as potential biomarkers for cancer diagnosis or therapeutic targets for cancer treatment.
    [Show full text]
  • Characterization of the Scavenger Cell Proteome in Mouse and Rat Liver
    Biol. Chem. 2021; 402(9): 1073–1085 Martha Paluschinski, Cheng Jun Jin, Natalia Qvartskhava, Boris Görg, Marianne Wammers, Judith Lang, Karl Lang, Gereon Poschmann, Kai Stühler and Dieter Häussinger* Characterization of the scavenger cell proteome in mouse and rat liver + https://doi.org/10.1515/hsz-2021-0123 The data suggest that the population of perivenous GS Received January 25, 2021; accepted July 4, 2021; scavenger cells is heterogeneous and not uniform as previ- published online July 30, 2021 ously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration. Abstract: The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly Keywords: glutaminase; glutamine synthetase; liver specialized hepatocyte subpopulations like glutamine syn- zonation; proteomics; scavenger cells. thetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been char- acterized extensively regarding expression of other genes and Introduction potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and There is a sophisticated structural-functional organization in perivenous GS-expressing hepatocytes from mouse and rat. the liver acinus with regard to ammonium and glutamine Apart from established markers of GS+ hepatocytes such as metabolism (Frieg et al. 2021; Gebhardt and Mecke 1983; glutamate/aspartate transporter II (GLT1) or ammonium Häussinger 1983, 1990). Periportal hepatocytes express en- transporter Rh type B (RhBG), we identified novel scavenger zymes required for urea synthesis such as the rate-controlling cell-specific proteins like basal transcription factor 3 (BTF3) enzyme carbamoylphosphate synthetase 1 (CPS1) and liver- and heat-shock protein 25 (HSP25).
    [Show full text]
  • Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice
    pharmaceutics Review Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice Malavika Deodhar 1, Sweilem B Al Rihani 1 , Meghan J. Arwood 1, Lucy Darakjian 1, Pamela Dow 1 , Jacques Turgeon 1,2 and Veronique Michaud 1,2,* 1 Tabula Rasa HealthCare Precision Pharmacotherapy Research and Development Institute, Orlando, FL 32827, USA; [email protected] (M.D.); [email protected] (S.B.A.R.); [email protected] (M.J.A.); [email protected] (L.D.); [email protected] (P.D.); [email protected] (J.T.) 2 Faculty of Pharmacy, Université de Montréal, Montreal, QC H3C 3J7, Canada * Correspondence: [email protected]; Tel.: +1-856-938-8697 Received: 5 August 2020; Accepted: 31 August 2020; Published: 4 September 2020 Abstract: In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition (competitive, non-competitive, mechanism-based) have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity.
    [Show full text]
  • Resolution of Carbon Metabolism and Sulfur-Oxidation Pathways of Metallosphaera Cuprina Ar-4 Via Comparative Proteomics
    JOURNAL OF PROTEOMICS 109 (2014) 276– 289 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot Resolution of carbon metabolism and sulfur-oxidation pathways of Metallosphaera cuprina Ar-4 via comparative proteomics Cheng-Ying Jianga, Li-Jun Liua, Xu Guoa, Xiao-Yan Youa, Shuang-Jiang Liua,c,⁎, Ansgar Poetschb,⁎⁎ aState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China bPlant Biochemistry, Ruhr University Bochum, Bochum, Germany cEnvrionmental Microbiology and Biotechnology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China ARTICLE INFO ABSTRACT Article history: Metallosphaera cuprina is able to grow either heterotrophically on organics or autotrophically Received 16 March 2014 on CO2 with reduced sulfur compounds as electron donor. These traits endowed the species Accepted 6 July 2014 desirable for application in biomining. In order to obtain a global overview of physiological Available online 14 July 2014 adaptations on the proteome level, proteomes of cytoplasmic and membrane fractions from cells grown autotrophically on CO2 plus sulfur or heterotrophically on yeast extract Keywords: were compared. 169 proteins were found to change their abundance depending on growth Quantitative proteomics condition. The proteins with increased abundance under autotrophic growth displayed Bioleaching candidate enzymes/proteins of M. cuprina for fixing CO2 through the previously identified Autotrophy 3-hydroxypropionate/4-hydroxybutyrate cycle and for oxidizing elemental sulfur as energy Heterotrophy source. The main enzymes/proteins involved in semi- and non-phosphorylating Entner– Industrial microbiology Doudoroff (ED) pathway and TCA cycle were less abundant under autotrophic growth. Also Extremophile some transporter proteins and proteins of amino acid metabolism changed their abundances, suggesting pivotal roles for growth under the respective conditions.
    [Show full text]
  • Spring 2013 Lecture 13-14
    CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna INTRODUCTION TO ENZYMES • Enzymes are usually proteins (some RNA) • In general, names end with suffix “ase” • Enzymes are catalysts – increase the rate of a reaction – not consumed by the reaction – act repeatedly to increase the rate of reactions – Enzymes are often very “specific” – promote only 1 particular reaction – Reactants also called “substrates” of enzyme catalyst rate enhancement non-enzymatic (Pd) 102-104 fold enzymatic up to 1020 fold • How much is 1020 fold? catalyst time for reaction yes 1 second no 3 x 1012 years • 3 x 1012 years is 500 times the age of the earth! Carbonic Anhydrase Tissues ! + CO2 +H2O HCO3− +H "Lungs and Kidney 107 rate enhancement Facilitates the transfer of carbon dioxide from tissues to blood and from blood to alveolar air Many enzyme names end in –ase 89 CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna Why Enzymes? • Accelerate and control the rates of vitally important biochemical reactions • Greater reaction specificity • Milder reaction conditions • Capacity for regulation • Enzymes are the agents of metabolic function. • Metabolites have many potential pathways • Enzymes make the desired one most favorable • Enzymes are necessary for life to exist – otherwise reactions would occur too slowly for a metabolizing organis • Enzymes DO NOT change the equilibrium constant of a reaction (accelerates the rates of the forward and reverse reactions equally) • Enzymes DO NOT alter the standard free energy change, (ΔG°) of a reaction 1. ΔG° = amount of energy consumed or liberated in the reaction 2.
    [Show full text]
  • Product Sheet Info
    Master Clone List for NR-19279 ® Vibrio cholerae Gateway Clone Set, Recombinant in Escherichia coli, Plates 1-46 Catalog No. NR-19279 Table 1: Vibrio cholerae Gateway® Clones, Plate 1 (NR-19679) Clone ID Well ORF Locus ID Symbol Product Accession Position Length Number 174071 A02 367 VC2271 ribD riboflavin-specific deaminase NP_231902.1 174346 A03 336 VC1877 lpxK tetraacyldisaccharide 4`-kinase NP_231511.1 174354 A04 342 VC0953 holA DNA polymerase III, delta subunit NP_230600.1 174115 A05 388 VC2085 sucC succinyl-CoA synthase, beta subunit NP_231717.1 174310 A06 506 VC2400 murC UDP-N-acetylmuramate--alanine ligase NP_232030.1 174523 A07 132 VC0644 rbfA ribosome-binding factor A NP_230293.2 174632 A08 322 VC0681 ribF riboflavin kinase-FMN adenylyltransferase NP_230330.1 174930 A09 433 VC0720 phoR histidine protein kinase PhoR NP_230369.1 174953 A10 206 VC1178 conserved hypothetical protein NP_230823.1 174976 A11 213 VC2358 hypothetical protein NP_231988.1 174898 A12 369 VC0154 trmA tRNA (uracil-5-)-methyltransferase NP_229811.1 174059 B01 73 VC2098 hypothetical protein NP_231730.1 174075 B02 82 VC0561 rpsP ribosomal protein S16 NP_230212.1 174087 B03 378 VC1843 cydB-1 cytochrome d ubiquinol oxidase, subunit II NP_231477.1 174099 B04 383 VC1798 eha eha protein NP_231433.1 174294 B05 494 VC0763 GTP-binding protein NP_230412.1 174311 B06 314 VC2183 prsA ribose-phosphate pyrophosphokinase NP_231814.1 174603 B07 108 VC0675 thyA thymidylate synthase NP_230324.1 174474 B08 466 VC1297 asnS asparaginyl-tRNA synthetase NP_230942.2 174933 B09 198
    [Show full text]
  • A Mathematical Model of Glutathione Metabolism Michael C Reed*1, Rachel L Thomas1, Jovana Pavisic1,2, S Jill James3, Cornelia M Ulrich4 and H Frederik Nijhout2
    Theoretical Biology and Medical Modelling BioMed Central Research Open Access A mathematical model of glutathione metabolism Michael C Reed*1, Rachel L Thomas1, Jovana Pavisic1,2, S Jill James3, Cornelia M Ulrich4 and H Frederik Nijhout2 Address: 1Department of Mathematics, Duke University, Durham, NC 27708, USA, 2Department of Biology, Duke University, Durham, NC 27708, USA, 3Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AK 72205, USA and 4Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA Email: Michael C Reed* - [email protected]; Rachel L Thomas - [email protected]; Jovana Pavisic - [email protected]; S Jill James - [email protected]; Cornelia M Ulrich - [email protected]; H Frederik Nijhout - [email protected] * Corresponding author Published: 28 April 2008 Received: 27 November 2007 Accepted: 28 April 2008 Theoretical Biology and Medical Modelling 2008, 5:8 doi:10.1186/1742-4682-5-8 This article is available from: http://www.tbiomed.com/content/5/1/8 © 2008 Reed et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Glutathione (GSH) plays an important role in anti-oxidant defense and detoxification reactions. It is primarily synthesized in the liver by the transsulfuration pathway and exported to provide precursors for in situ GSH synthesis by other tissues. Deficits in glutathione have been implicated in aging and a host of diseases including Alzheimer's disease, Parkinson's disease, cardiovascular disease, cancer, Down syndrome and autism.
    [Show full text]
  • Lecture 11 - Biosynthesis of Amino Acids
    Lecture 11 - Biosynthesis of Amino Acids Chem 454: Regulatory Mechanisms in Biochemistry University of Wisconsin-Eau Claire 1 Introduction Biosynthetic pathways for amino acids, Text nucleotides and lipids are very old Biosynthetic (anabolic) pathways share common intermediates with the degradative (catabolic) pathways. The amino acids are the building blocks for proteins and other nitrogen-containing compounds 2 2 Introduction Nitrogen Fixation Text Reducing atmospheric N2 to NH3 Amino acid biosynthesis pathways Regulation of amino acid biosynthesis. Amino acids as precursors to other biological molecules. e.g., Nucleotides and porphoryns 3 3 Introduction Nitrogen fixation is carried out by a few Text select anaerobic micororganisms The carbon backbones for amino acids come from glycolysis, the citric acid cycle and the pentose phosphate pathway. The L–stereochemistry is enforced by transamination of α–keto acids 4 4 1. Nitrogen Fixation Microorganisms use ATP and ferredoxin to Text reduce atmospheric nitrogen to ammonia. 60% of nitrogen fixation is done by these microorganisms 15% of nitrogen fixation is done by lighting and UV radiation. 25% by industrial processes Fritz Habers (500°C, 300!atm) N2 + 3 H2 2 N2 5 5 1. Nitrogen Fixation Enzyme has both a reductase and a Text nitrogenase activity. 6 6 1.1 The Reductase (Fe protein) Contains a 4Fe-4S Text center Hydrolysis of ATP causes a conformational change that aids the transfer of the electrons to the nitrogenase domain (MoFe protein) 7 7 1.1 The Nitrogenase (MoFe Protein) The nitrogenase Text component is an α2β2 tetramer (240#kD) Electrons enter the P-cluster 8 8 1.1 The Nitrogenase (MoFe Protein) An Iron-Molybdenum cofactor for the Text nitrogenase binds and reduces the atmospheric nitrogen.
    [Show full text]
  • Characterization and Improved Properties of Glutamine Synthetase from Providencia Vermicola by Site-Directed Mutagenesis
    www.nature.com/scientificreports OPEN Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site- Received: 21 November 2017 Accepted: 19 June 2018 directed mutagenesis Published: xx xx xxxx Wu Zuo2, Leitong Nie2, Ram Baskaran2, Ashok Kumar3 & Ziduo Liu1,2 In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed signifcant increase in the activity and stabilities at a wide range of temperatures. The Km values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine −5 were 15.7 ± 1.1, (25.2 ± 1.5) × 10 and 32.6 ± 1.7 mM, and the kcat were 17.0 ± 0.6, 9.14 ± 0.12 and 30.5 ± 1.0 s−1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efciency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50 °C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its fexibility due to the breaking of hydrogen bonds between product and enzyme.
    [Show full text]