Studies on the structure and function of pyruvate dehydrogenase complexes

R. de Abreu

Research output: Thesisinternal PhD, WU

Abstract

The aim of the present investigation was to obtain more information of the structure and function of the pyruvate dehydrogenase complexes from <em>Azotobacter vinelandii</em> and <em>Escherichia coli.</em><p/>In chapter 2 a survey is given of the recent literature on pyruvate dehydrogenase complexes.<p/>In chapter 3 results are presented, describing the behaviour of the pyruvate dehydrogenase complexes from <em>A.vinelandii</em> and <em>E.coli</em> Crookes on blue dextran Sepharose 4B columns. It is shown that the 4-component pyruvate dehydrogenase complex from <em>A.vinelandii</em> binds strongly to the column through its low-mol.wt. transacetylase while the pyruvate dehydrogenase complex from <em>E.coli</em> Crookes does not bind. The pyruvate dehydrogenase complex from <em>A.vinelandii</em> can be eluted with 0.6 M potassium chloride as an active 3-component complex. Properties of the 3-component complex, such as the Hill coefficient of the overall reaction, the stimulation by AMP and the inhibition by acetyl-CoA are not different from the original complex. The 3-component complex, however, shows a 3-fold increase of lipoamide dehydrogenase activity which is much larger than the increase in FAD content.<p/>In chapter 4, the results from studies with radioactive pyruvate and radioactive N-ethymaleimide are presented. The results from these studies show that maximally four radioactive groups per mole FAD are incorporated into the high-mol.wt. lipoyltransacetylase component of the pyruvate dehydrogenase complex from <em>A.vinelandii</em> and to the lipoyltransacetylase component of the pyruvate dehydrogenase complex from <em>E.coli</em> when the complexes are incubated with [2- <sup><font size="-1">14</font></SUP>C] pyruvate, magnesium chloride and TPP or with N-ethyl [2,3- <sup><font size="-1">14</font></SUP>C] maleimide in the presence of pyruvate, magnesium chloride and TPP under anaerobic conditions. With 10 mM [2- <sup><font size="-1">14</font></SUP>C] pyruvate, the low-mol.wt. transacetylase is also labelled; to this enzyme three to four [ <sup><font size="-1">14</font></SUP>C] acetylgroups are bound. The 3-component pyruvate dehydrogenase complex from <em>A.vinelandii,</em> eluted by chromatography from a blue dextran- Sepharose 4B column, binds a maximum of four [ <sup><font size="-1">14</font></SUP>C] acetyl groups per mole of FAD. From these results it is concluded, that four lipoyl groups per mole of FAD are present in the complexes from both sources. Furthermore possible stoichiometries of the different enzyme components are discussed.<p/>In chapter 5, the crosslinking studies of bifunctional reagents with the pyruvate dehydrogenase complexes from <em>A.vinelandii</em> and <em>E.coli</em> are described. The results from these studies show that lipoamide dehydrogenase and high-mol.wt. transacetylase, as well as that low-mol.wt. transacetylase and pyruvate dehydrogenase are at close distances in the 4-component complex from <em>A.vinelandii.</em> In the 3-component pyruvate dehydrogenase complex from <em>A.vinelandii</em> and the pyruvate dehydrogenase complex from <em>E.coli,</em> all three components seem to be organized at close distances to one another. Crosslinking with diimidates in the presence of pyruvate, Mg <sup><font size="-1">2+</font></SUP>and TPP show a conformational change of the transacetylase core of the pyruvate dehydrogenase complex from <em>A.vinelandii.</em> In the complex from <em>E.coli</em> no crosslinking was observed under these conditions between the lipoyltransacetylase monomers but rather close positioning was observed of lipoyl groups from the lipoyltransacetylase component and pyruvate dehydrogenase component which indicate that SH-groups are exposed on the pyruvate dehydrogenase in the presence of pyruvate Mg <sup><font size="-1">2+</font></SUP>and TPP.<p/>In chapter 6 results are presented of immuno-chemical studies on the pyruvate dehydrogenase complexes from <em>A.vinelandii</em> and <em>E.coli.</em> The results of the immunochemical studies indicate that the pyruvate dehydrogenase component is located at the surface of both complexes. In addition, the low-mol.wt. lipoyltransacetylase is also located at the surface of the 4-component pyruvate dehydrogenase complex from <em>A.vinelandii.</em> The low-mol.wt. lipoyltransacetylase seems to protect the 4-component complex against inactivation by its antiserum. Furthermore it is shown that the pyruvate dehydrogenase complexes from <em>A.vinelandii</em> and <em>E.coli</em> are serologically different.<p/>The results from the studies presented in this thesis seem to fit with the generally accepted model of the pyruvate dehydrogenase complex from <em>E.coli</em> presented by Reed and coworkers (chapter 2). The pyruvate dehydrogenase complex from <em>A.vinelandii.</em> seems to consist of a "core" of high-molecular weight transacetylase chains to which a lipoamide dehydrogenase dimer is bound. At the surface of the complex pyruvate dehydrogenase components and low-molecular weight transacetylase are bound.<p/>It is apparent that more experiments are needed to be performed, to obtain a more detailed model of especially the pyruvate dehydrogenase complex from <em>A.vinelandii.</em><p/><em></em>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Veeger, C., Promotor
Award date13 Dec 1978
Place of PublicationWageningen
Publisher
Publication statusPublished - 1978

Keywords

  • azotobacter
  • biochemistry
  • escherichia coli
  • metabolism
  • microorganisms
  • oxidoreductases
  • synthesis
  • chemical analysis
  • exposure
  • environmental degradation
  • kinetics
  • ecotoxicology

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