title: Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis.
abstract: Acetohydroxyacid synthase 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[2-14C]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 14C 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. We 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.
authors.forename: P M
authors.initials: PM
authors.lastname: Silverman
authors.forename: L
authors.initials: L
authors.lastname: Eoyang
mesh_terms: D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006885:Hydroxybutyrates / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007773:Lactates / Q000096:biosynthesis*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D006885:Hydroxybutyrates; D007527:Isoenzymes; D007773:Lactates; D011773:Pyruvates; C006359:alpha-acetolactate; C017092:bromopyruvate; D000095:Acetolactate Synthase; D007652:Oxo-Acid-Lyases