Interchain acetyl transfer in the E2 component of bacterial pyruvate dehydrogenase suggests a model with different roles for each chain in a trimer of the homooligomeric component.

The bacterial pyruvate dehydrogenase complex carries out conversion of pyruvate to acetyl-coenzyme A with the assistance of thiamin diphosphate (ThDP), several other cofactors, and three principal protein components, E1-E3, each present in multiple copies. The E2 component forms the core of the complexes, each copy consisting of variable numbers of lipoyl domains (LDs, lipoic acid covalently amidated at a lysine residue), peripheral subunit binding domains (PSBDs), and catalytic (or core) domains (CDs). The reaction starts with a ThDP-dependent decarboxylation on E1 to an enamine/C2α̃ carbanion, followed by oxidation and acetyl transfer to form S-acetyldihydrolipoamide E2, and then transfer of this acetyl group from the LD to coenzyme A on the CD. The dihydrolipoamide E2 is finally reoxidized by the E3 component. This report investigates whether the acetyl group is passed from the LD to the CD in an intra- or interchain reaction. Using an Escherichia coli E2 component having a single LD, two types of constructs were prepared: one with a Lys to Ala substitution in the LD at the Lys carrying the lipoic acid, making E2 incompetent toward post-translational ligation of lipoic acid and, hence, toward reductive acetylation, and the other in which the His believed to catalyze the transthiolacetylation in the CD is substituted with A or C, the absence of His rendering it incompetent toward acetyl-CoA formation. Both kinetic evidence and mass spectrometric evidence support interchain transfer of the acetyl groups, providing a novel model for the presence of multiples of three chains in all E2 components, and their assembly in bacterial enzymes.