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

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid syn thesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate compo nents: biotin carboxylase, carboxyltransferase, and the biotin carboxyl car rier 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 superfamil y of enzymes. Included in this superfamily are D-Ala D-Ala ligase, glutathi one synthetase, carbamyl phosphate synthetase, N-5-carboxyaminoimidazole ri bonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of h ighly conserved residues between them. Four of these residues of biotin car boxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-di rected mutagenesis studies based on their structural homology with conserve d residues of other ATP-grasp enzymes. These mutants were subjected to kine tic analysis to characterize their roles in substrate binding and catalysis , In all four mutants, the K-m value for ATP was significantly increased, i mplicating 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 a nd cocrystallized ADP or nucleotide analogs. In addition, the maximal veloc ity 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 h ave misaligned the reactants for optimal catalysis.