STRUCTURAL STUDIES OF NUCLEOTIDE DEPENDENT ENZYMES

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Nucleotides serve important roles in virtually all biochemical processes where they provide building blocks for DNA and RNA, act as currencies of energy in various metabolic pathways, serve as modulatory os enzyme activity, and function as transient electron acceptors and donors. The objective of this research program is to understand on a structural level the role of nucleotides in the overall enzymatic mechanisms of three specific protein systems: acetyl CoA carboxylase which is Mg2+-ATP dependent, kanamycin nucleotidyltransferase which can employ ATP, GTP, or UTP as a substrate, and UDP-galactose 4-epimerase which requires NAD+ for activity. For the proposed studies, a combination of site-directed mutagenesis experiments, x-ray crystallography, and kinetic measurements will be employed. These protein systems were chosen for study because they have been well characterized by both biochemical and biophysical techniques, the amount of protein necessary for the proposed studies is readily isolated from over-expression systems in Escherichia coli, and crystals suitable for x-ray analyses have been obtained. In addition, these proteins play important metabolic roles that are relevant to health-related issues as discussed below. Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids by converting acetyl-CoA to malonyl- CoA. The production of malonyl-CoA is also one of the rate-controlling reactions for this biochemical pathway. Impairment of fatty acid synthesis can lead to significant cardio-vascular problems. Consequently, the proposed studies of acetyl CoA carboxylase are critically important for understanding normal fatty acid metabolism. Kanamycin nucleotidyltransferase catalyzes the inactivation of kanamycin, an aminoglycoside antibiotic used in the treatment of infections due to aerobic Gram-negative bacteria. The structural studies proposed in this grant application will provide three-dimensional information concerning the active site of this enzyme and may ultimately lead to the design of aminoglycoside antibiotics that are resistant to attack by the enzyme. Finally, UDP-galactose 4-epimerase, the third enzyme in the Leloir pathway which converts galactose to glucose, plays a major role in proper galactose metabolism. Deficiencies in this metabolic cycle can lead to various diseased states including galactosemia.