Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.
The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptible to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the--AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH--group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.
D000107:Acetylation; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D008063:Thioctic Acid / Q000378:metabolism*
D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D005033:Ethylmaleimide