1972 — 1981 |
Fromm, Herbert |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mechanism and Control of Enzyme Action |
0.915 |
1981 — 2003 |
Fromm, Herbert Honzatko, Richard (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Studies On the Mechanism and Control of Enzyme Action
9985565 Fromm Adenylosuccinate synthetase, a ubiquitous enzyme, participates in two important metabolic pathways, the purine nucleotide cycle and the de novo biosynthesis of adenine nucleotides. The former maintains ATP levels in active muscle tissue, whereas the latter provides essential precursors for RNA and DNA biosynthesis. Even in organisms that lack a pathway for de novo purine biosynthesis, adenylosuccinate synthetase still plays a central role in salvage mechanisms for adenine nucleotides. This research seeks a detailed understanding of the structure-function relationships of the synthetases from Escherichia coli and from mouse. Of central interest is the monomer-dimer subunit equilibrium of adenylosuccinate synthetases and its effect on enzyme activity. Does IMP, one of the substrates of the synthetase, induce a transition from inactive monomers to active synthetase dimers, and if so, is this phenomenon a universal mechanism of regulation for all synthetases? In addition, the mechanism by which stringent effectors influence activity of the E. coli enzyme is unsettled. The stringent effect is a response by E. coli to conditions of stress, such as starvation, resulting in elevated levels of guanine nucleotides, which putatively inhibit adenylosuccinate synthetase and many other enzymes. Is inhibition by some stringent effectors enhanced by the chemical action of adenylosuccinate synthetase, and do stringent effectors inhibit the synthetase by a single mechanism? The synthetase-catalyzed reaction is second order with respect to the essential metal cation, yet in crystal structures only one Mg2+ is present, associated with GDP. The second Mg2+ binds putatively to L-aspartate. Experiments will be designed to test whether the a-carboxyl group of L-aspartate is an essential recognition element for the binding of the second Mg2+. The synthetase catalyzes its overall reaction as a sequence of two partial reactions. Experiments will determine whether the synthesis of 6-phosphoryl-IMP (first reaction) or the synthesis of adenylosuccinate (second reaction) is rate limiting. The proposed research will employ a variety of techniques in physical biochemistry, including X-ray crystallography, 31P NMR, analytical ultracentrifugation, and kinetics. The studies will employ recombinant E. coli and/or mouse synthetases, expressed in and isolated from E. coli. Specific mutants of the E. coli and mouse synthetases will be constructed in order to test several structure-function hypotheses.
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0.915 |
1985 |
Fromm, Herbert Jerome |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Brain and Tumor Phosphotransferases
How enzymes function as catalysts and how they are regulated at the molecular and hormonal level is of fundamental importance to the biologist. The studies proposed in this application involve the investigation of enzymes involved in glycolysis and gluconeogenesis and their regulation. In the case of glycolysis, experiments will be centered around two isozymes, brain and muscle hexokinase. The activity of the latter phosphotransferase has been shown to decrease in streptozotocin-induced diabetes and returns to normal upon the administration of insulin. We have shown that the rate of degradation of muscle hexokinase in the diabetic rat is three times greater than in the normal animal. We propose to investigate the rates of synthesis and degradation in insulin treated diabetic animals and the rate of synthesis of muscle hexokinase in normal and diabetic animals. Brain hexokinase is the pacemaker of glycolysis in the brain and red blood cells, and its activity is controlled by its products and inorganic phosphate. We plan to investigate the mechanism of brain hexokinase regulation using kinetic and nuclear magnetic resonance techniques. The gluconeogenic enzyme we propose to investigate is fructose 1,6-bisphosphatase. It has recently been demonstrated that fructose 2,6-bisphosphate is a potent inhibitor of the phosphatase and an activator of the glycolytic enzyme, phosphofructokinase. We have preliminary evidence showing how the inhibitor affects the activity of fructose 1,6-bisphosphatase at the molecular level and we plan to pursue these investigations using nuclear magnetic resonance procedures. In addition, we expect to investigate the kinetic mechanism of action of the enzyme responsible for the biosynthesis of the inhibitor, fructose-6-P 2 kinase, and finally, we propose to study its regulation using kinetic protocols. The same procedures will be used to investigate the mechanism and regulation of fructose 1,6-bisphosphatase.
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1 |
1986 — 1991 |
Fromm, Herbert Jerome |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism and Regulation of Brain and Liver Phosphatases
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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1992 — 2007 |
Fromm, Herbert Jerome |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Regulatory Enzymes of Glycolysis and Gluconeogenesis
Proposed investigations focus on fructose-1,6-bisphosphatase, an enzyme of gluconeogenesis subject to coordinate regulation by metabolites, and hexokinase isoforms I and II, enzymes that inhibit apoptosis through their association with mitochondria. A proposed model describes how small and localized ionformational change, induced by the binding of allosteric or active site inhibitors, triggers global conformation change in fructose-1,6-bisphosphatase. Directed mutations, the formation of hybrid tetramers by subunit exchange, fluorescence spectroscopy, kinetics, and structure determinations by x-ray diffraction, test the validity of the proposed model in accounting for positive cooperativity in allosteric inhibition, and iynergism between allosteric and active-site directed inhibitors. Research will define new sites from which an appropriate ligand can reinforce the action of physiological inhibitors of fructose-1,6-bisphosphatase, leading in the long run to new drugs that ameliorate high levels of serum glucose. The release of hexokinase isoforms I and II from mitochondria is a key step in apoptosis. Experiments here are designed to reveal the mechanism of release of hexokinase isoforms from the mitochondrion, and the subunit structure of hexokinase isoforms in their mitochondrion-bound states. Mitochondrial binding and release properties of mutant forms of hexokinase types I and II will test specifc mechanisms of ligand-induced release. Studies of spin-labeled hexokinase bound to reconsituted vesicles, using electron paramagnetic resonance methods, will determine whether the enzyme exists on the mitochondrion as a multimer, and if so the relative arrangement of subunits in that multimer. Investigations of fructose-1,6-bisphosphatase are of direct significance to the development of drugs that reduce high levels of serum glucose associated with diabetes type II. Work on hexokinase isoforms I and II may provide new strategies in triggering apoptosis (cell death) in cancers.
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