Self-assembly and Catalytic Activity of the Pyruvate Dehydrogenase Multienzyme Complex of "Escherichia Coli" PDF Download
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Author: David L. Bates
Publisher:
ISBN:
Category :
Languages : en
Pages : 4
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Author: David L. Bates
Publisher:
ISBN:
Category :
Languages : en
Pages : 4
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Author: Maria E. Maldonado
Publisher:
ISBN:
Category :
Languages : en
Pages : 6
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Author: Jaeyoung Song
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 160
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Book Description
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: Stephen A. Kuby
Publisher: CRC Press
ISBN: 9780849369889
Category : Medical
Languages : en
Pages : 646
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Book Description
This comprehensive monograph consists of two parts: Volume I, entitled Enzyme Catalysis, Kinetics, and Substrate Binding; and Volume II, entitled Mechanism of Enzyme Action. Volume I focuses on several aspects of enzyme catalytic behavior, their steady-state and transient-state kinetics, and the thermodynamic properties of substrate binding. Packed with figures, tables, schemes, and photographs, this volume contains over 1,000 references, including references regarding enzymology's fascinating history. This comprehensive book is of particular interest to enzymology students, teachers, and researchers. Volume II presents selected "cutting edge" examples of techniques and approaches being pursued in biochemistry. This up-to-date resource includes 11 chapters, which illustrate important theoretical and practical aspects of enzyme mechanisms. It also features selected examples in which today's most important techniques, ideas, and theories are used to elaborate on the intricate nature of enzyme action mechanisms. This particular volume provides important information for both the novice and the seasoned investigator.
Author: Steven Ken Akiyama
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 450
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Author: Alasdair Steven
Publisher: Garland Science
ISBN: 1000064905
Category : Medical
Languages : en
Pages : 881
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Book Description
Molecular Biology of Assemblies and Machines provides a comprehensive narrative of the ways in which macromolecular structures assemble and how they interact with other complexes and organelles in the cell. Richly illustrated in full color, the text is written for advanced undergraduates, graduate students, and researchers in biochemistry, molecular biology, biophysics, cell biology, chemistry, structural biology, immunology, microbiology, and medicine.
Author: Anand Balakrishnan
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 235
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Book Description
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: Ruth Porter
Publisher: John Wiley & Sons
ISBN: 0470718447
Category : Science
Languages : en
Pages : 192
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Book Description
The Novartis Foundation Series is a popular collection of the proceedings from Novartis Foundation Symposia, in which groups of leading scientists from a range of topics across biology, chemistry and medicine assembled to present papers and discuss results. The Novartis Foundation, originally known as the Ciba Foundation, is well known to scientists and clinicians around the world.
Author: Frank Jordan
Publisher: CRC Press
ISBN: 0824758714
Category : Medical
Languages : en
Pages : 632
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Book Description
Thiamine: Catalytic Mechanisms in Normal and Disease States brings together the most recent developments in thiamine diphosphate (TDP)-requiring enzyme research and details the mechanisms of catalysis and structure-function relationships, as well as pathophysiological aspects of a spectrum of diseases associated with TDP-requiring enzymes. Providing new insights into neurogenerative diseases, this volume associates defects in the function of TDP-dependent enzymes with numerous metabolic disorders and disease states and offers novel aspects of thiamine enzymes in chiral synthesis as well as new perspectives on the cellular role of thiamine triphosphate and thiamine triphosphates.
Author: R. Holland Cheng
Publisher: World Scientific
ISBN: 9789812386151
Category : Science
Languages : en
Pages : 440
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Book Description
Electronic version of the text of the same title with additional audio and video links.
