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 (PDHc) carries out conversion of pyruvate to acetyl-coenzymeA with the assistance of three principal protein components E1, E2 and E3(1) (Scheme 1). In the mammalian enzyme(2), there are three additional components mostly responsible for regulation: a kinase, a phosphatase and an E3-binding protein (E3BP)(3, 4). Each component exists in multiple copies with total molecular masses in the range of 4.5–10 MDa. The multi-domain E2 component forms the core of the complexes [see domain structure for E2 from Escheerichia coli (E2ec) in Scheme 2]. It consists of variable number (1–3) of LD’s to which the cofactor lipoic acid is covalently attached in a post-translational reaction, a PSBD, and a CD, where acetyl-CoA is produced(1). Although the number of copies of the icosahedral mammalian E2 is still controversial, there is agreement that the sum (E2+E3BP) is 60, a multiple of three(5–7), while there are 24 copies of the octahedral E. coli E2 (E2ec), again a multiple of three(1). For the E. coli PDHc a stoichiometry of 12 E1ec dimers, 8 E2ec trimers and 6 E3ec dimers was proposed(8–12). All 24 copies of E2ec have LD and CD, and it is plausible that inter-chain acyl transfer would take place between an LD of one chain and the CD of a different one, with mobility of LD afforded by flexible Ala-Pro-rich linkers(8–11, 13–16). The comment in ref. 12, that “A morphological unit consisting of three subunits appears to be important in the assembly of both types of polyhedral forms” (icosahedral or octahedral), prompted us to explore potential mechanistic consequences of this observation. Scheme 1 Mechanism of pyruvate dehydrogenase complex Scheme 2 Domain architecture of wild-type E2ec. We here address the question of whether the acetyl group is passed from the LD to the CD in an intra- or inter-chain reaction by using an E. coli E2 component designed with only a single LD (1-lip E2ec), rather than the three LDs in the wild type enzyme (3-lip E2ec). Earlier, it was shown that the activities of 1-lip E2ec and 3-lip E2ec are virtually the same, while the 1-lip E2ec offers advantages for mechanistic studies(17–19). Two types of constructs of the 1-lip E2ec were prepared to test our hypothesis: one in which the lysine on the LD ordinarily carrying the lipoic acid is changed to alanine (K41A), and two others in which the histidine believed to catalyze the trans-thiolacetylation in the CD is substituted to A or C, H399C and H399A. The first variant is incompetent towards post-translational ligation of the lipoic acid, hence towards reductive acetylation. The other two variants are potentially incompetent towards acetyl-CoA formation by virtue of the absence of the catalytic histidine residue. These constructs then enable us to carry out a complementation experiment: should acetyl transfer reaction proceed intra-chain, the two types of constructs should each be inactive either together or individually; however, should acetyl transfer proceed inter-chain, addition of the two types of constructs should produce measurable activity. As control, the K41A/H399C doubly substituted variant was also constructed, carrying neither a lipoylation site, nor the active center His. The experimental design for this complementation of E2ec variants is illustrated in Figure 1. We confirmed the results of the kinetics experiments by ascertaining acetylCoA formation from CoA using mass spectrometric analysis. The results suggest a plausible model for utilization of multiples of three chains present in E2 components, and for their assembly in the bacterial class of such complexes. Figure 1 Complementation experiments designed to test for inter-chain acetyl transfer on E2ec. (a) The experiment to monitor inter-subunit acetyl transfer between K41A E2ec and H399C E2ec. (b) Control experiment with doubly substituted variant (K41A/H399C E2ec). ...