Determination of the Chain Stoichiometries from the Number of Reactive Sulfhydryl Groups in the Pyruvate Dehydrogenase Complexes of Azotobacter vinelandii and Escherichia coli

The pyruvate dehydrogenase complex from Azotobacter vinelandii incubated with 0.05–0.7 mM [2-14C]pyruvate, magnesium chloride and thiamine pyrophosphate under anaerobic conditions at 0°C, incorporates four [14C]acetyl groups per mole FAD which are bound to the high-molecular-weight lipoyl transacetylase. With 10 mM pyruvate, the low-molecular-weight lipoyl transacetylase is also labelled; to this enzyme 3–4 [14C]acetyl groups are bound per mole FAD. This enzyme is not labelled when pyruvate is used in concentrations lower than 0.7 mM. The complex of A. vinelandii pre-labelled with non-radioactive N-ethylmaleimide did not incorporate label onto low-molecular-weight lipoyl transacetylase; a maximum of four acetyl groups are bound to the high-molecular weight lipoyl transacetylase. Maximum incorporation of four [14C]acetyl groups per mole FAD is found, within five seconds, on the lipoyl transacetylase component when the Escherichia coli complex is labelled under anaerobic conditions, also in the presence of 10 mM pyruvate. In both complexes all incorporated acetyl groups are bound to sulfhydryl groups since they can be completely removed with hydroxylamine, by performic acid oxidation and by reaction with coenzyme A and arsenite. Under aerobic conditions a slow deacetylation is observed, which is blocked in the presence of N-ethylmaleimide. The multi-enzyme complex from A. vinelandii pre-labelled with non-radioactive N-ethylmaleimide and then labelled with N-ethyl [2,3-14C]maleimide in the presence of pyruvate, magnesium chloride and thiamine pyrophosphate incorporates under anaerobic conditions at 0°C, even at 10 mM pyruvate, a maximum of four N-ethyl[14C]maleimide groups per mole of FAD. The label is almost exclusively bound to the high-molecular-weight lipoyl transacetylase of the A. vinelandii complex. The E. coli complex binds only 2–3 N-ethyl[14C]maleimide groups per mole of FAD under these conditions. Direct labelling and correction for labelling without pyruvate yields 4–5 N-ethyl-[14C]maleimide per mole FAD. The low-molecular-weight lipoyl transacetylase can be removed by chromatography of the complex from A. vinelandii on a blue-dextran–Sepharose 4B column [De Abreu et al. (1977) FEBS Lett. 82, 89–92]. The complex eluted from the column, consisting of the other three enzyme components, binds a maximum of four [14C]acetyl groups per mole of FAD, even with 10 mM pyruvate. It is concluded that the lipoyl/FAD ratio is four in the complexes as isolated by us from both sources. The interpretation of this ratio with respect to the stoichiometries of the different enzyme components will be discussed.