Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-Å resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the threedimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-Å resolution.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.

When Q-Sepharose was used in the purification of the V nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed that resulted in a separation of the nitrogenase component 1 protein (Av1') into two forms, labeled Av1'A and Av1'B. Even though both forms possessed the same enzymatic behavior, Av1'A exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1'B. Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms, with Av1'A possessing a trimer (alpha beta 2) pattern compared to the more typical tetramer (alpha 2 beta 2) pattern found for Av1'B. Metal analysis indicated a V-to-Fe ratio of 1:19 for Av1'A and 1:15 (or 2:30) for Av1'B, while acid-labile sulfide analysis showed that Av1'A possessed about half as much sulfide as Av1'B. EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations, with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that Av1'A is an incomplete form of the VFe protein, containing only one cofactor and one P cluster with an additional [Fe4-S4]-like cluster. The presence of a V storage protein in A. vinelandii was also investigated. Although no V storage protein was found, two V-binding proteins were observed.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K) biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K) biocytin profile and 7.5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1 proton of biocytin.The first committed step in fatty acid biosynthesis is catalyzed by acetyl-CoA carboxylase (1). The enzyme is found in all animals, plants, and bacteria and catalyzes the biotin-dependent carboxylation of acetyl-CoA to form malonyl-CoA in the two steps of Reaction 1.In Escherichia coli the enzyme is composed of three components: biotin carboxylase, carboxyltransferase, and biotin carboxyl carrier protein, which contains the biotin covalently attached to the ⑀-nitrogen of lysine (2). Biotin carboxylase catalyzes the first half-reaction in which biotin is carboxylated to form carboxybiotin. Carboxyltransferase catalyzes the second half-reaction where the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. Both biotin carboxylase and the carrier protein are homodimers, whereas the carboxyltransferase component is an ␣ 2 ␤ 2 tetramer. Each of the three components can be isolated separately, and the biotin carboxylase and carboxyltransferase components retain catalytic activity in the absence of the other two components. Moreover, biotin carboxylase and carboxyltransferase will utilize free biotin as a substrate, which makes them ideal model systems for studying mechanistic aspects of biotin-dependent enzymes.Most mechanistic studies of acetyl-CoA carboxylase have focused on the biotin carboxylase component because the gene for the enzyme has bee...

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...