Doctor of Philosophy (PhD)


Biological Sciences

Document Type



Acetyl-CoA carboxylase catalyzes the first step in the synthesis 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. This enzyme uses the cofactor biotin as a carboxyl carrier. In order for the carboxylation of biotin to occur, biotin must be deprotonated at its N-1 position. It has been proposed that the active site residues cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin. To test this hypothesis, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the Km for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. However, the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin. A bisubstrate analog inhibitor of carboxyltransferase was synthesized by covalently linking biotin to Coenzyme A via an acyl bridge between the sulfur of Coenzyme A and the N-1 of biotin. The inhibitor was found to have an inhibition constant of 23 ± 2 ìM, which means it binds the enzyme 350-times tighter than biotin. The bisubstrate analog demonstrated competitive inhibition versus malonyl-CoA and noncompetitive inhibition versus biocytin. This is consistent with an ordered kinetic mechanism with malonyl-CoA binding first. A precursor to the inhibitor, chloroacylated biotin, was capable of inhibiting the differentiation of 3T3-L1 cells in a dose-dependent manner. Treatment with chloroacylated biotin resulted in a decrease in acetyl-CoA carboxylase activity and inhibited lipid accumulation. Our results support recent studies that indicate acetyl-CoA carboxylase may be a suitable target as an anti-obesity therapeutic.



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Committee Chair

Grover L. Waldrop