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 sitedirected 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.Acetyl-CoA carboxylase catalyzes the first committed step in long chain fatty acid synthesis, namely the formation of malonyl-CoA from acetyl-CoA, MgATP, and bicarbonate. Found in all plants, animals, and bacteria, this enzyme is biotin-dependent, with the following two-step reaction mechanism (1).Enzyme-biotin-CO 2The Escherichia coli form of this enzyme consists of three separable components. The biotin carboxylase component catalyzes the first half-reaction, which involves the phosphorylation of bicarbonate to form a carboxyphosphate intermediate, followed by the transfer of the carboxyl group to the 1Ј nitrogen of biotin (2). The carboxyltransferase component catalyzes the second half-reaction. In vivo the biotin molecule is linked to the biotin carboxyl carrier protein through an amide bond to a specific lysine residue. Both biotin carboxylase and carboxyltransferase retain activity in the absence of the other two components and will also use free biotin as a substrate (3). The crystal structure of the biotin carboxylase component has been solved and is the only three-dimensional structure of a biotindependent carboxylase, making it the paradigm for structurefunction analysis of this class of enzymes (4). Two years after the solution of the crystal structure, Artymiu...