Doctor of Philosophy (PhD)


Biological Sciences

Document Type



Acetyl-CoA carboxylase is an essential enzyme in all plants, animals, and bacterie, where it catalyzes the committed step in fatty acid synthesis. The Escherichia coli version of the enzyme has three functional components that dissociate readily: biotin carboxylase, carboxyltransferase, and a biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin, using bicarbonate as the carboxylate source. Biotin carboxylase is a member of a functionally diverse superfamily of proteins known as the ATP-grasp enzymes. In the first study, four residues of biotin carboxylase, Lys 116, Lys 159, His 209, and Glu 276, were selected for site-directed mutagenesis based on their structural homology and strict conservation among the ATP-grasp enzymes. The resulting mutants were subject to kinetic characterization. The Km for ATP for all four mutants was significantly elevated relative to the wild type enzyme, implicating the residues in binding ATP. The Vmax for the biotin-dependent ATPase reaction was 30- to 260-fold lower than wild type, suggesting that the mutations have misaligned the substrates for optimal catalysis. In the second study, three more biotin carboxylase mutants were made based on their homology to naturally occurring mutations of human biotin-dependent carboxylases. The three mutations result in metabolic diseases, indicating that the residues involved are functionally important. The mutants M169K, R338Q, and R338S were constructed and characterized. The two Arg mutants displayed uncoupling of biotin carboxylation from ATP hydrolysis, suggesting that the residue Arg 338 is important for aligning the carboxyphosphate intermediate for optimal carboxyl transfer to biotin. Interestingly, all three mutants displayed negative cooperativity with respect to bicarbonate, suggesting a communication between the two subunits of biotin carboxylase. Additionally, the level of residual biotin-dependent ATPase activity for M169K and R338S was consistent with the severity of the phenotypes of the patients carrying the corresponding mutations, thus establishing a molecular basis for the diseases.



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Committee Chair

Grover Waldrop