Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.
The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.
D001152:Arsenicals / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D001152:Arsenicals; D003432:Cross-Linking Reagents; D011768:Pyruvate Dehydrogenase Complex; C031672:4-((bromoacetyl)amino)phenyl arsenoxide; D008058:Dihydrolipoamide Dehydrogenase