Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate

Journal Article · Tue Dec 12 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00451a007· OSTI ID:6855470

Flournoy, D S; Frey, P A ^[1]

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1. Univ. of Wisconsin, Madison (USA)

The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenease component (E{sub 1}) 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 E{sub 1} component are similar. Pyruvate protects the PDH complex and the E{sub 1} component against inactivation by fluoropyruvate. Dihydrolipoamide protects the E{sub 1} component from inactivation. TPP is not covalently bound to the PDH complex or to the E{sub 1} component by the inactivating reaction. When ({sup 14}C)fluoropyruvate is used to inactivate the PDH complex, {sup 14}C remains bound to the complex after gel filtration. This bound radioactivity is cleaved from the protein by NH{sub 2}OH, {sup {minus}}OH, and NaBH{sub 4} but not by dilute acid. When released by {sup {minus}}OH, greater than 90% of the {sup 14}C cochromatographs with acetate on DEAE-Sephadex. When released by NaBH{sub 4}, and {sup 14}C is recovered as ({sup 14}C)ethanol. Colorimetric analysis for sulfhydryl groups on the native E{sub 1} component and the inactivated E{sub 1} component, using 5,5{prime}-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 {plus minus} 0.03 sulfhydryl residues. Fluoropyruvate is known to generate acetyl-TPP at the active of E{sub 1}. The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E{sub 1} component inactivates the enzyme.

• OSTI ID: 6855470
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:25; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
AMIDES
AMINO ACIDS
BACTERIA
BIOLOGICAL EFFECTS
CARBON 14 COMPOUNDS
CARBONIC ACID DERIVATIVES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
CYSTEINE
ENZYMES
ESCHERICHIA COLI
ETHANOL
FLUORINATED ALIPHATIC HYDROCARBONS
HALOGENATED ALIPHATIC HYDROCARBONS
HEMIACETAL DEHYDROGENASES
HYDROXY COMPOUNDS
INACTIVATION
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC FLUORINE COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
SEPARATION PROCESSES
THIOLS
TRACER TECHNIQUES
UREA