Site-directed mutagenesis of ATP binding residues of biotin carboxylase. Insight into the mechanism of catalysis

J Biol Chem. 2001 Jul 6;276(27):24991-6. doi: 10.1074/jbc.M101472200. Epub 2001 May 9.


Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N(5)-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K(m) value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / biosynthesis
  • Adenosine Triphosphate / metabolism*
  • Amino Acid Substitution
  • Bicarbonates / metabolism
  • Biotin / metabolism
  • Carbon-Nitrogen Ligases / genetics
  • Carbon-Nitrogen Ligases / metabolism*
  • Catalysis
  • Crystallography, X-Ray
  • Escherichia coli
  • Kinetics
  • Magnesium / metabolism
  • Mutagenesis, Site-Directed
  • Protein Conformation


  • Bicarbonates
  • Biotin
  • Adenosine Triphosphate
  • Carbon-Nitrogen Ligases
  • biotin carboxylase
  • Magnesium