Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehyrogenase multienzyme complex of Escherichia coli

Abstract

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.

Spectroscopic characterization of thioredoxin covalently modified with monofunctional organoarsenical reagents

“…Reduction of a disulfide bond may yield spatially close thiols that are readily modified covalently by trivalent organoarsenical reagents of the form R-As=0 (or R-AsX2), where R is an organic group and X is a halide ion (Stocken & Thompson, 1949;Stevenson et al, 1978). These reagents have been applied to enzyme systems such as 2-ketoacid de-hydrogenase multienzyme complexes (Stevenson et al, 1978; Adamson Adamson et al, 1984Adamson et al, , 1986, lipoamide dehydrogenase (Danson et al, 1986), mitochondrial coupling factors (Sanaoi, 1982), transport systems (Frost & Lane, 1985), pyridine nucleotide transhydrogenase (Voordouw et al, 1981), and adenylate cyclase (Drummond, 1981). These reagents form stable cyclic dithioarsenites that are readily reversed on the addition of small molecular weight dithiol reagents such as 2,3-dimercaptopropanol (Whittaker, 1947;Stocken & Thompson, 1946).…”

“…We also wanted to determine whether or not, upon modification, there would be a perturbation of the secondary and tertiary structure of thioredoxin. As evaluation of the effect of chemical modification on the structure of thioredoxin is a necessary prerequisite for studies involving thioredoxin chemically modified with a bifunctional reagent (e.g., BrCH2CONHPhAsO) such r-S-, that this form of thioredoxin (thioredoxin As-L~S~' PhNHCOCH2Br) could function as an active-site-directed inactivator for thioredoxin reductase and ribonucleotide reductase in a manner similar to pyruvate dehydrogenase multienzyme complex (Adamson et al, 1984(Adamson et al, , 1986Holmes & Stevenson,86).5 µ In this study we have monitored the inhibition of thioredoxin by measuring the loss of activity and Experimental Procedures Materials 2,3-Dimercaptopropanol was obtained from Sigma Chemical Co. Mercaptoethylamine and 5,5/-dithiobis(2-nitrobenzoic acid) were purchased from Pierce Chemical Co. L-Tryptophan and L-tyrosine were from Calbiochem. 1,4-Dibromobutane and arsenic trioxide were obtained from Aldrich Chemical.…”