Studies on "Escherichia Coli" Pyruvate Dehydrogenase Complex. I. Effect of Bromopyruvate on the Catalytic Activities of the Complex PDF Download
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Author: Maria E. Maldonado
Publisher:
ISBN:
Category :
Languages : en
Pages : 6
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Author: Maria E. Maldonado
Publisher:
ISBN:
Category :
Languages : en
Pages : 6
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Author: Claire Ann CaJacob
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 416
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Author: David L. Bates
Publisher:
ISBN:
Category :
Languages : en
Pages : 4
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Author: Hong-Wu He
Publisher: Springer
ISBN: 3662444313
Category : Science
Languages : en
Pages : 471
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This book presents essential research on a class of environmentally friendly alkylphosphonate herbicides. This class of herbicides acted as a competitive inhibitor of the pyruvate dehydrogenase complex (PDHc) to control weeds. The bioreasoning and systematic approach, from basic research to field tests of candidate compounds, are introduced. The basic research covers the molecular design, chemical synthesis, biological activities evaluation, structure-activity relationship analysis and structural optimization. Subsequently, the book reviews the biochemistry of PDHc inhibitors, the selectivity between mammals and plants, and the mechanism of herbicidal activity of novel alkylphosphonates as selective PDHc inhibitors. Field trials for selected alkylphosphonate candidates as herbicides are also included. This book provides a sound basis for the rational design and development of novel herbicides as effective PDHc inhibitors with good enzyme-selective inhibition of plant PDHc between mammals and plants. These studies take full advantages of the low toxicity and low residual impact of selective PHDc inhibitors to design an effective and environmentally friendly herbicide. This book is based on twenty years of research on alkylphosphonates and phosphorus-containing PDHc inhibitors, and demonstrates how to develop these PDHc inhibitors as an effective and “green” herbicide candidate. Hong-Wu He, PhD, is a Professor at the Key Laboratory of Pesticide & Chemical Biology, Ministry of Education of China, and Director of the Institute of Pesticide Chemistry, College of Chemistry, Central China Normal University, China. Hao Peng, PhD, and Xiao-Song Tan are both Associate Professors at the Key Laboratory of Pesticide & Chemical Biology, Ministry of Education of China, College of Chemistry, Central China Normal University, China.
Author: Marilyn A. Apfel
Publisher:
ISBN:
Category :
Languages : en
Pages : 5
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Author: Jaeyoung Song
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 160
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The pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli (E. coli) is the best characterized of the 2-oxoacid dehydrogenase complexes. The complex plays a role as catalyst for the conversion of pyruvate to acetyl Coenzyme A (acetylCoA) by three enzyme components in the complex. The complex is comprised of 24 copies of the dimeric pyruvate dehydrogenase (E1ec; 99,474 Da), a cubic core of 24 copies of dihydrolipoamide acetyltransferase (E2ec; 65,959 Da), and 12 copies of dihydrolipoamide dehydrogenase (E3ec; 50,554 Da) (1-3). The crystal structure of the E. coli pyruvate dehydrogenase complex E1 subunit (E1ec) has been deterimined, and there were three missing regions (residues 1-55, 401-413, and 541-557) remaining absent in the model due to high flexibilities of these regions (4). Most bacterial pyruvate dehydrogenase complexes from either Gram-positive or Gram-negative bacteria have E1 components with an a2 homodimeric quaternary structure. In a sequel to our previous publications (5-8), the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance was presented. In the study, sequence-specific NMR assignments for six residues in the N-terminal 1-55 region, and for a glycine in each of the two mobile active center loops of the E1 component, a 200 kDa homodimer was made. This was accomplished by using site-specific substitutions and appropriate labeling patterns, along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, the spectra were also examined in the presence of: (a) a reaction intermediate analog known to affect the mobility of the active center loops, (b) an E2 component construct consisting of a lipoyl domain (LD) and peripheral subunit binding domain (PSBD) and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain, and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component. Among three components of PDHc, E2ec consists of 24 chains, and in the overall reaction, the lipoyl domain is reductively acetylated by E1ec and pyruvate, and S-acetyldihydrolipoyl domain transfers acetyl group to Coenzyme A leading acetylcoenzyme A production. Even though the precise number of E2 subunits is still ambiguous, in many cases including human PHDc (9), the sum is a multiple of three chains indicating that multiples of chains can affect the acetyl transfer to CoA by interchain acetyl transfer. To answer this question, the E2 component from E. coli specifically designed with only a single lipoyl domain (LD, 1-lip E2ec), rather than the three lipoyl domains found in the wild type enzyme (3-lip E2ec) was used. Earlier, it was shown that the activities of these two enzymes are virtually the same, while the 1-lip E2ec provides obvious advantages for mechanistic studies (10). At the same time, it is also important to point out that there indeed are other sources of the E2 component with only a single domain, such as from Mycobacterium tuberculosis. To study the question, two constructs of the 1-lip E2ec were prepared, one in which the lysine on the LD ordinarily carrying the lipoic acid is changed to alanine (henceforth K41A), and a second one in which the histidine believed to catalyze the transacetylation in the catalytic domain (CD) is substituted to A or C, H399C and H399A. The first is incompetent towards posttranslational ligation of the lipoic acid, hence towards reductive acetylation. The second one is incompetent towards acetylCoA formation, by virtue of the absence of the catalytic histidine residue. This is a biochemical version of a classical crossover experiment, as should the reaction proceed within one chain the two constructs should each be inactive either together or individually. On the other hand, should the reaction proceed by an interchain mechanism, addition of the two constructs should produce measurable activity. Both kinetic and mass spectrometric evidence supported the second scenario. Hence, plausible model/explanation for the multiples of three chains present in each E2 component as well as for their assembly was suggested.
Author: Edith R. Schwartz
Publisher:
ISBN:
Category :
Languages : en
Pages : 6
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Author: Kenneth L. Kirk
Publisher: Springer Science & Business Media
ISBN: 1475746059
Category : Medical
Languages : en
Pages : 374
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Biochemistry of Halogenated Organic Compounds has been written as a general reference source for researchers in several related areas, including organic chemists, medicinal chemists, pharmacologists, toxicologists, and medical researchers. The development of halogenated compounds as medicinal agents and pharmacological tools and the fascinating biochemi cal processes that have been discovered and studied using these analogues have generated extremely active areas of research and an enormous volume of literature. Thus, halogenated organic compounds pervade every aspect of biochemistry, a fact made apparent by the numerous reviews and monographs available on individual topics-halogenated nucleosides, halogenated carbohydrates, and so forth. Given the quantity of material already written on these topics, some of which material is quite current, it might be asked whether a one-volume review of these subjects is useful, or possible. Having now completed this work, I feel the answer to both questions is an emphatic yes. There are fascinating stories to be related in each area, and, where appropriate, I have attempted to develop these topics . from a historical perspective. For example, the discovery of the anticancer activity of fluorouracil, the unraveling of the several mechanisms of its action, and the development of a host of later generations of anticancer and antiviral agents based on the parent fluoro-, iodo-, bromo-, and trifluoromethylpyrimidines were, and are, contributions of major magnitude to medical science.
Author: Anand Balakrishnan
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 235
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Spectroscopic identification and characterization of covalent and non-covalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. While nuclear magnetic resonance (NMR) methods which provide detailed chemical insights have been successfully employed previously, limited examples are available in the literature for large enzyme complexes. Enzymes utilizing cofactors provide promising examples for such studies when synthetic routes to labeled cofactor analogs and protocols for reconstitution of apo-enzymes with such analogs are readily available. Syntheses of key isotope enriched thiamin diphosphate (ThDP) analogs -- [C2, C6' -- 13C2] ThDP, [N4' -- 15N]ThDP and [C2 -- 13C]ThDP -- enabled first detection of (i) various ionization/tautomerization states of ThDP during the catalytic cycle of three ThDP dependent enzymes using cross polarization magic angle spinning (CPMAS) solid state NMR (SSNMR) spectroscopy and (ii) [C2, C6' -- 13C2] ThDP covalent intermediates on the E1 component (E1p) during the catalytic cycle of E. coli pyruvate dehydrogenase multi-enzyme complex (PDHc) by filter experiments including solution 1-D 1H-13C HSQC NMR. Direct evidence was gathered for the 4'-aminopyrimidinium form (APH+) on ThDP molecules bound to (i) S. cerevisiae yeast pyruvate decarboxylase (YPDC) (ii) E1p and (iii) the E1 component of E. coli 2-oxoglutarate dehydrogenase complex (E1o) using 13C and 15N CPMAS SSNMR. The thiazolium C2-H bond was found to be slightly acidic in the cofactor bound to these enzymes. 15N SSNMR experiments confirmed the formation of the 1', 4'-iminopyrimidine tautomer in presence of substrate analogs; a mechanism is proposed for the stabilization of this biologically rare tautomer in enzyme active-sites. Using rapid chemical quench in conjunction with solution NMR, pre-steady state analyses were performed on the native PDHc and PDH complexes reconstituted with E1p active-site loop variants of very low PDHc activity. The C2-[alpha]-lactylThDP intermediate could not be detected under any of the conditions used, indicating that its formation is slower than its decarboxylation. The enamine intermediate accumulates at a rate 110 s-1 on E1p and PDHc, while the rates are 100-fold slower for the PDHc variants. 2-acetylThDP could be detected on E1p only during its reaction with pyruvate and the artificial electron acceptor DCPIP. Reductive acetylation of the lipoyl domain in a pre-steady state single turn-over experiment (a model for the E1p-E2p reductive acetyl transfer reaction) was determined by mass spectrometry. Combined, these kinetic results from artificial oxidation reactions suggest the enamine is very well stabilized by E1p and oxidation of the enamine and substrate channeling to E2p are favored by intact PDHc. These studies provide unprecedented insight into the acid-base and covalent electrophilic roles of ThDP in enzyme catalysis and the methods described herein are applicable to all such complexes.
Author: Peter N. Lowe
Publisher:
ISBN:
Category :
Languages : en
Pages : 7
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