How does the pentose phosphate pathway generate NADPH?
• The pentose phosphate pathway reduces 2 moles of NADP⁺ to 2 moles of NADPH + H⁺ for each mole of glucose-6-P that is oxidatively decarboxylated to generate 1 mole of ribulose-5-P.
• NADPH functions as a strong reductant in anabolic pathways (that is, ones that synthesize biomolecules from smaller precursors) and in detoxification reactions that neutralize reactive oxygen species. NAD⁺ is primarily used as an oxidant in catabolic pathways (that is, ones that release energy).
• The pentose phosphate pathway is divided into the oxidative phase, which generates NADPH, and the nonoxidative phase, which interconverts sugar phosphates to regenerate glucose-6-P by using transketolase and transaldolase enzymes.
• Flux through the pentose phosphate pathway is regulated to meet three distinct metabolic states of the cell: (1) a need for NADPH; (2) a need to replenish nucleotide pools; and (3) a need to generate ATP from glucose-6-P.
• Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the commitment step in the pentose phosphate pathway and is highly regulated by the NADP⁺/NADPH concentration ratio to control flux through the pathway; NADP⁺ stimulates enzyme activity to increase NADPH levels in the cell.
• Mutations in the human G6PD gene result in reduced intracellular levels of NADPH, which decreases the amount of reduced glutathione needed to protect cells from oxidative stress.
• The antimalarial drug primaquine and the natural product vicine found in fava beans induce oxidative stress that can be deleterious to individuals who are G6PD deficient. The geographic overlap between the prevalence of G6PD deficiencies in human populations and high rates of malaria provide supporting evidence that G6PD deficiencies are protective against malarial death.
How are gluconeogenesis and glycolysis reciprocally regulated to control flux?
• Gluconeogenesis synthesizes glucose from noncarbohydrate sources when dietary glucose is limiting and glucose stores have been depleted. Much of the glucose used by the brain and red blood cells in humans comes from gluconeogenesis occurring in liver and kidney cells.
• Gluconeogenesis and glycolysis share seven enzymes, catalyzing reversible reactions in both pathways. The four solely gluconeogenic enzymes are pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase (FBPase-1), and glucose-6-phosphatase, which bypass three exergonic reactions in glycolysis (catalyzed by pyruvate kinase, phosphofructokinase-1, and hexokinase).
• Pyruvate carboxylase and phosphoenolpyruvate carboxykinase catalyze the pyruvate kinase bypass reactions by converting pyruvate to phosphoenolpyruvate, using phosphoryl transfer energy from ATP (pyruvate carboxylase reaction) and GTP (phosphoenolpyruvate carboxykinase reaction).
• FBPase-1 catalyzes the phosphofructokinase-1 (PFK-1) bypass reaction by converting fructose-1,6-BP to fructose-6-P. FBPase-1 and PFK-1 are reciprocally regulated by the allosteric effectors AMP, fructose-2,6-BP, and citrate in response to energy charge and hormonal signaling.
• The hexokinase reaction in glycolysis is bypassed by the gluconeogenic enzyme glucose-6-phosphatase, which is localized to the ER lumen and thus physically isolated from the hexokinase reaction in the cytosol.
• Hormonal control of PFK-1 and FBPase-1 is mediated in liver cells by fructose-2,6-BP. Fructose-2,6-BP is structurally related to fructose-6-P and fructose-1,6-BP but is not a metabolic intermediate in either the glycolytic or gluconeogenic pathways.
• The apparent Km of PFK-1 for its substrate fructose-6-P is decreased 25-fold by fructose-2,6-BP levels, whereas fructose-2,6-BP increases the apparent Km of FBPase-1 for its substrate fructose-1,6-BP by 15-fold. The net effect is that high levels of fructose-2,6-BP stimulate flux through the glycolytic pathway and inhibit flux through the gluconeogenic pathway.
• The level of fructose-2,6-BP in liver cells is controlled by the dual-function enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which is hormonally regulated by phosphorylation.
• Insulin signaling stimulates dephosphorylation of PFK-2/FBPase-2, leading to higher levels of fructose-2,6-BP and activation of flux through the glycolytic pathway; glucagon signaling decreases fructose-2,6-BP levels and stimulates gluconeogenesis.
• The Cori cycle uses gluconeogenesis in liver cells to convert lactate produced by muscle cells into glucose, thereby replenishing glucose levels and supporting muscle contraction. The energy cost of running gluconeogenesis (liver) and glycolysis (muscle) at the same time is net 4 ATP equivalents, which is the difference between 2 ATP produced by anaerobic glycolysis and 4 ATP + 2 GTP consumed by gluconeogenesis.
What are the enzymes and reactions of glycogen degradation and synthesis?
• Glycogen is a storage form of glucose in animals and consists of branched homopolysaccharides linked by α(1→4) and α(1→6) glycosidic bonds. Glycogen degradation and synthesis occur in the cytosol, with the substrate for these reactions being the free ends (nonreducing ends) of the branched polymer.
• The four key enzymes required for reversible degradation and synthesis of glycogen are glycogen phosphorylase, glycogen synthase, and the glycogen branching and debranching enzymes.
• Glycogen phosphorylase catalyzes a reaction that releases glucose-1-P from glycogen in a phosphorolysis reaction involving inorganic phosphate and cleavage of the α(1→4) glycosidic bond.
• Glucose-1-P is converted to glucose-6-P, which can be used for glycolysis in muscle cells or dephosphorylated in liver cells and exported to other tissues. Glycogen phosphorylase activity is stimulated by glucagon and epinephrine signaling.
• Glycogen synthase is activated by insulin signaling and adds glucose to the nonreducing ends in a reaction requiring UDP-glucose. Glycogen synthase uses the bond energy available in UDP-glucose to form α(1→4) glycosidic bonds at the nonreducing ends of the glycogen particle.
• Branching and debranching enzymes modify glycogen complexes to facilitate glycogen degradation (debranching) and glycogen synthesis (branching) through the cleavage and formation of α(1→6) glycosidic bonds.
• Glycogen degradation and synthesis require the enzyme phosphoglucomutase, which interconverts glucose-1-P and glucose-6-P through the formation of a bisphosphorylated enzyme intermediate.
How is glycogen metabolism regulated by hormonal signaling?
• Glycogen phosphorylase exists in the active R-state conformation and the inactive T-state conformation. The equilibrium between the R state and T state is shifted by metabolite allosteric control (rapid response) and by hormone regulated phosphorylation/dephosphorylation (delayed response).
• The enzyme phosphorylase kinase phosphorylates glycogen phosphorylase and is a downstream target of glucagon and epinephrine signaling in liver cells.
• Allosteric activation of unphosphorylated muscle glycogen phosphorylase by AMP binding induces the R-state conformation, whereas ATP and glucose-6-P are allosteric inhibitors of unphosphorylated muscle glycogen phosphorylase by competing with AMP for binding to the allosteric site.
• Phosphorylated liver glycogen phosphorylase is allosterically inhibited by glucose binding, which shifts the equilibrium from the R state to the T state and facilitates dephosphorylation of the enzyme in response to insulin signaling.
• Glycogen synthase adds glucose to the nonreducing ends of glycogen by using the nucleotide sugar UDP-glucose. The enzyme UDP-glucose pyrophosphorylase catalyzes a reaction involving the attack of a phosphoryl oxygen from glucose-1-P on the α phosphate of UTP to generate UDP-glucose.
• Glycogenin is both an anchor protein for the glycogen core particle and an enzyme that catalyzes the glycosyltransferase and synthesis reactions needed to generate the initial glycogen chain.
• Glucagon signaling stimulates protein kinase A (PKA) activity, leading to net phosphorylation of glycogen phosphorylase and glycogen synthase, which results in increased glycogen degradation and glucose release.
• Akt phosphorylates a serine residue (S1) on the muscle phosphatase regulatory protein GM, which stimulates its regulatory activity, thereby leading to the recruitment and activation of protein phosphatase 1. The net result is that glycogen degradation is inhibited and glycogen synthesis is stimulated.
• Epinephrine signaling results in protein kinase A–mediated phosphorylation of the GM subunit on a different serine residue (S2), which stimulates the dissociation of protein phosphatase 1 from GM and the subsequent inhibition of protein phosphatase 1 activity. The net result is that glycogen synthesis is inhibited and glycogen degradation is stimulated.
What is the molecular basis for the most common glycogen storage diseases?
• von Gierke disease, Hers disease, and Pompe disease are prevalent human glycogen storage diseases. von Gierke disease is due to a deficiency in the enzyme glucose-6-phosphatase; Hers disease is a defect in the liver enzyme glycogen phosphorylase; and Pompe disease is due to a deficiency in lysosomal α-1,4-glucosidase, also called acid maltase.
• Three less common human glycogen storage diseases are McArdle disease (a defect in muscle glycogen phosphorylase), Cori disease (a defect in glycogen debranching enzyme), and Andersen disease (a defect in glycogen branching enzyme).
• Individuals with Pompe disease can be treated by enzyme replacement therapy, which requires weekly infusions of purified recombinant lysosomal α-1,4-glucosidase protein. Enzyme replacement therapy does not cure the disease, but it can slow its progression.
Everyday example: CO-17: G6PD Mutations Cause Favism
Everyday BioChem 