The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Does a unifying model exist for the complexes from gram-negative bacteria?

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15-19 U/mg and an overall yield of 40%. The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 +/- 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains. The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5-1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45-0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2. As shown before [Bosma, H.J., de Kok, A., Markwijk, B.W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273-280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociated into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components. A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.