Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis
Acetohyroxyacid synthease I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo(2-/sup 14/C)pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of /sup 14/C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. The authors confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.
- Research Organization:
- Albert Einstein College of Medicine, Bronx, NY
- OSTI ID:
- 6501645
- Journal Information:
- J. Bacteriol.; (United States), Vol. 169:6
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BUTYRIC ACID
BIOSYNTHESIS
LACTIC ACID
LIGASES
ALKYLATION
ENZYME ACTIVITY
CARBON 14 COMPOUNDS
ELECTROPHORESIS
ESCHERICHIA COLI
POLYPEPTIDES
PYRUVIC ACID
STOICHIOMETRY
TRACER TECHNIQUES
BACTERIA
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ENZYMES
HYDROXY ACIDS
ISOTOPE APPLICATIONS
KETO ACIDS
LABELLED COMPOUNDS
MICROORGANISMS
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
PROTEINS
SYNTHESIS
550201* - Biochemistry- Tracer Techniques