The long-term objective of this application is to determine how fructose 1,6-bisphosphatase (FBPase), a key regulatory enzyme in gluconeogenesis, functions as a catalyst and is controlled at the molecular level. Experiments are to be conducted with the enzyme from mammalian brain and liver tissue. Although brain is not a gluconeogenic tissue, it does contain FBPase. Because the substrate for this enzyme is the product of a key step in brain glycolysis involving the enzyme phosphofructokinase, coordinated regulation of these two enzymes is essential to brain tissue viability. Brain FBPase is somewhat unique among mammalian FBPases for two reasons: it does not require exogenous metal ion for activity, and it is not inhibited by AMP. One of the aims of this application is to investigate the regulation of the brain enzyme as well as its mechanism of action. The role of exogenous metal ion in FBPase enzymology is not clearly understood. Attempts will be made to evaluate the role of exogenous metal in catalysis and regulation from kinetic experiments in the nonphysiological direction in the presence and absence of AMP. Liver FBPase has two or three tightly associated metal ions per monomeric subunit. The enzyme itself is a tetramer. The function of these metals will be investigated using a variety of techniques, including nuclear magnetic resonance spectroscopy. The metals may facilitate catalysis by binding to the oxygen atoms at the 1-phosphate position of the substrate, thereby aiding nucleophilic attack by a hydroxyl ion on the phosphorous atom. On the other hand, they may simply tether the substrate to the enzyme by hydrogen bonding a liganded water molecule to the substrate, or they may merely provide the enzyme with the proper conformation for catalysis. Answers to these questions may be provided by replacing the natural metal Zn2+, with the paramagnetic ion Mn2+. By taking advantage of the fact that paramagnetic ions can effectively relax diamagnetic nuclei, and that this effect is distance dependent, it is possible to measure the distance from the metal binding site to a particular nucleus, such as the 1-P of the substrate. A knowledge of these distances may provide insights into the role of the metals in catalysis. It is also possible using this technique to obtain information on how physiologically important regulators, such as AMP and fructose 2,6-bisphosphatate, function at the molecular level to inhibit FBPase.
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