Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate.
The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenase component (E1) are rapidly inactivated by low concentrations of fluoropyruvate in a thiamin pyrophosphate (TPP) dependent process. The inactivation rates for the PDH complex and for its E1 component are similar. Pyruvate protects the PDH complex and the E1 component against inactivation by fluoropyruvate. Dihydrolipoamide protects the E1 component from inactivation. TPP is not covalently bound to the PDH complex or to the E1 component by the inactivating reaction. When [14C]fluoropyruvate is used to inactivate the PDH complex, 14C remains bound to the complex after gel filtration. This bound radioactivity is cleaved from the protein by NH2OH, -OH, and NaBH4 but not by dilute acid. When released by -OH, greater than 90% of the 14C cochromatographs with acetate on DEAE-Sephadex. When released by NaBH4, and 14C is recovered as [14C]ethanol. Colorimetric analysis for sulfhydryl groups on the native E1 component and the inactivated E1 component, using 5,5'-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 +/- 0.03 sulfhydryl residues. Fluoropyruvate is known to generate acetyl-TPP at the active site of E1. The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E1 component inactivates the enzyme.
Flournoy
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Institute for Enzyme Research, Graduate School, College of Agricultural and Life Sciences, University of Wisconsin, Madison 23705.
Frey
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D002384:Catalysis; D003545:Cysteine; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology*; D013835:Thiamine Pyrophosphate / Q000493:pharmacokinetics / Q000031:analogs & derivatives; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; C017319:3-fluoropyruvate; C051074:2-acetylthiamine pyrophosphate; C007409:dihydrolipoamide; D008063:Thioctic Acid; D003545:Cysteine; D013835:Thiamine Pyrophosphate