Studies on 2-oxoacid dehydrogenase multienzyme complexes of Azotobacter vinelandii

In this thesis, some studies on the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Azotobacter vinelandii are described; the emphasis strongly lies on the pyruvate dehydrogenase complex.A survey of the literature on 2-oxoacid dehydrogenase complexes is given in chapter 1. It appears that the A.vinelandii pyruvate dehydrogenase complex resembles the complexes from other gram-negative bacteria with respect to its composition and working mechanism. The A.vinelandii complex is however much smaller than the pyruvate dehydrogenase complexes isolated from other sources.Chapter 2 describes the procedure that has been optimized for the isolation of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes (PDC and OGDC respectively) from A.vinelandii. In comparison to the previous isolation procedure, several advantages exist. The A.vinelandii PDC is obtained as an essentially pure three-component complex, in a high yield (40-50%). 80% of the losses can be accounted for by discarded side-fractions, which indicates that the complex is hardly inactivated during its purification. The specific activity of the final preparation is about two times higher (15-19 U/mg) than previously could be obtained. From these observations we conclude that the formerly observed "fourth component" of A.vinelandii PDC was a mere contaminant. With the revised procedure, the 2-oxoglutarate dehydrogenase complex (OGDC) is obtained in a high yield (40-50%), free from contaminants. In the "old" procedure this complex was irreversibly inactivated by the action of protamine sulfate.In chapter 3 some observations on the A.vinelandii OGDC are reported. The molecular mass of this complex is of the order of 2.4 to 3.2 MDa, as determined by laser light-scattering measurements. The three component enzymes have the same molecular masses as have been reported for the OGDC's of Escherichia coli and pig-heart. The activity of the complex is regulated by its substrates in an analogues way as has been reported for the E.coli complex, and we therefore conclude that the A.vinelandii complex probably strongly resembles the OGDC of E.coli. In this chapter, an isolation procedure for the lipoamide dehydrogenase component is described, and it is shown that the lipoamide dehydrogenase components of the A.vinelandii PDC and OGDC probably are identical.The association behaviour of the A.vinelandii pyruvate dehydrogenase complex is described in chapter 4. From sedimentation and light-scattering studies we conclude that a monomer-dimer equilibrium exists for this complex; the molecular mass of the monomer has been estimated that 800 kDa. In this thesis, this monomer-dimer mixture is referred to as the 18 S form of the complex. Upon addition of polyethylene glycol 6000 and MgCl ² , the 18 S form of the complex aggregates into a large structure, resembling the pyruvate dehydrogenase complex of E.coli with respect to its sedimentation, coefficient (56 S) and its appearance on electron micrographs. The isolated dihydrolipoyl transacetylase component of A.vinelandii PDC has a molecular mass of 2 MDa, and on electron micrographs it resembles the dihydrolipoyl acetyltransferase component of E.coli. It is concluded that this large structure probably is composed of 32 subunits. Upon the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components, this large particle dissociates into the smaller structures that are characteristic for the intact A.vinelandii complex. The small (18 S) and the large (56 S) forms of the (sub)complexes are in slow equilibrium, and this equilibrium can be perturbed by high hydrostatic pressure. From light-scattering measurements at varying pressures it is concluded that the 56 S form of the complex probably is an octamer of the 800 kDa monomers.The measurements concerning the chain-stoichiometry of A.vinelandii PDC are described in chapter 5. A novel method for the determination of chain-ratios was developed, based on the covalent modification of lysine residues in the three component enzymes with trinitrobenzene sulfonic acid. With this technique, an average chain ratio of 1.3:1:0.5 (pyruvate hydrogenase: dihydrolipoyl acetyl transferase:lipoamide dehydrogenase) was found for the isolated A.vinelandii PDC. In combination with the results of chapter 4, it is concluded that A.vinelandii PDC is based on a tetrameric dihydrolipoyl acetyltransferase core, to which the periferal components are bound in a non-covalent way. The complex can be reconstituted from its individual components, and from these reconstitution experiments it follows that the complex has maximal activity when three pyruvate dehydrogenase dimers and one lipoamide dehydrogenase dimer are bound to the dihydrolipoyl transacetylase tetramer.In chapter 6, the results of acetylation experiments are given. It is shown that the reductive acetylation of the lipoyl groups probably is the rate-limiting step in the reaction sequence of the A.vinelandii pyruvate dehydrogenase complex. In so-called servicing experiments, an extensive exchange of acetyl groups between individual (monomeric) pyruvate dehydrogenase complex particles is found. This phenomenon (inter-core transacetylation) has until now only been observed for the A.vinetandii complex. It is shown that the inter-core transacetylation occurs when two monomeric particles are associated. Although the transacetylation reactions show large effects in the servicing experiments, these reactions are however too slow to be of physiological importance. The servicing experiments also show that the large " E.coli -like" isolated dihycirolipoyl acetyltransferase component is composed of rather independently operating tetramers, i.e. the large form of the A.vinelandii PDC does not function as a large entity.In chapter 7, the results of the three preceding chapters are summarized and translated into a three-dimensional model of the molecular organisation of the A.vinelandii PDC. The merits of this model are discussed in relation to the generally accepted model for the pyruvate dehydrogenase complex of E.coli. It is suggested that the pyruvate of Azotobacter vinelandii could represent the morphological subunit of the larger structure that is found in Escherichia coli and perhaps in other gramnegative bacteria. It is concluded that further experiments have to be performed, in which the complexes of the two organisms are directly compared. to establish whether such a unifying model does exist