Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthes is of long-chain fatty acids. The Escherichia coli form of the enzyme consi sts of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed m utagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from th e mutant form off the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino term inus of the mutant form of the enzyme and nickel affinity chromatography. T his system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to sev eral different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar res ults 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 K- m for biotin. The activity of the ATP synthesis reaction catalyzed by bioti n carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-f old by the mutations. The ATP synthesis reaction required biotin to stimula te the activity in the wild-type; however, biotin did not stimulate the act ivity of the mutant enzymes. The results showed that the mutations have abo lished 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 subs trate-induced synergism by biotin in biotin carboxylase.