Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermoph ilus was reconstituted in vitro from recombinant proteins derived from gene s over-expressed in Escherichia coli, Titrations of the icosahedral (60-mer ) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate dec arboxylase (E1, alpha(2)beta(2)) and dihydrolipoyl dehydrogenase (E3, alpha (2)) peripheral components indicated a variable composition defined predomi nantly by tight and mutually exclusive binding of E1 and E3 with the periph eral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E 1 and E3. and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of seco ndary interactions between the E1 and E3 subunits and E2 that only become a pparent on assembly. Exact geometrical distribution of E1 and E3 is unlikel y and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlat ion of the subunit composition with the overall catalytic activity of the e nzyme complex confirmed the lack of any requirement for precise stoichiomet ry or strict geometric arrangement of the three catalytic sites and emphasi zed the crucial importance of the flexibility associated with the lipoyl do mains and intramolecular acetyl group transfer in the mechanism of active-s ite coupling.