The long term objective of this research is to understand the catalytic mechanism of acetyl CoA carboxylase from E. coli. Acetyl CoA carboxylase catalyzes the biotin-dependent carboxylation of acetyl CoA to form malonyl CoA in the first step in fatty acid synthesis. The E. coli form of the enzyme consists of three components: (1) biotin carboxylase which transfers CO2 to biotin, (2) biotin carboxyl carrier protein which contains the biotin cofactor and (3) carboxyltransferase which transfers CO2 from biotin to acetyl CoA to make malonyl CoA. There are three specific aims of this proposal. (1) To determine the roles of active site residues in the catalytic mechanism of biotin carboxylase. The crystal structure of biotin carboxylase has been solved and refined to 1.8 A resolution and represents the first three-dimensional model of a biotin-dependent carboxylase. Three different sets of site-directed mutations will be done. First, residues Glu-211, Glu-288, Asn-290, and Arg-292 will be mutated to determine their role in the formation of the carboxyphosphate intermediate. Second, residues Cys-230 and Lys-238 will be mutated to determine if they act as an ion pair to remove the N1' proton of biotin prior to carboxylation. Third, glycine residues at positions 153, 163, 164, 166, and 168 will be mutated to determine if a domain of biotin carboxylase acts as a lid that closes down on the active site during catalysis. (2) A multi-substrate analog of biotin carboxylase will be synthesized and tested as an inhibitor. The multisubstrate inhibitor will be useful in crystallographic, kinetic, and binding studies. (3) While the determination of the three-dimensional structure of carboxyltransferase is underway, the multiple isotope effect method will be used to distinguish between a stepwise versus a concerted reaction. Mechanistic studies on biotin-dependent enzymes have been lacking. With the availability of a three-dimensional structure of biotin-dependent carboxylase the first structure-function studies are possible and should contribute to understanding the catalytic mechanism of this important class of enzymes.
