Biosynthesis of glucose – gluconeogenesis Carbohydrates provide a significant portion of human caloric intake

All cells are dependent on glucose. Glucose level in blood plasma must be stable. Brain is especially sensitive to the decrease of glucose level (the daily glucose requirement of the brain in a typical adult human being is about 120 g). Red blood cells use only glucose as a fuel. 160 g of glucose needed daily by the whole body. The amount of glucose present in body fluids is about 20 g, and that readily available from glycogen is approximately 190 g. During period of fasting glycogen in liver is mobilized but it only lasts 12 to 24 hours and this source of glucose may not fulfill metabolic need. During a longer period of starvation organism must synthesize glucose from smaller noncarbohydrate precursor molecules.

Liver and kidney are major sites of glucose synthesis Main precursors: lactate, pyruvate, glycerol and some amino acids Under fasting conditions, gluconeogenesis supplies almost all of the body’s glucose Gluconeogenesis – universal pathway. It present in animals, microorganisms, plants and fungi Plants synthesize glucose from CO 2 using the energy of sun, microorganisms – from acetate and propionate Gluconeogenesis – synthesis of glucose from noncarbohydrate precursors

Gluconeogenesis is not a Reversal Glycolysis 1. Glucose + ATP  glucose-6-phosphate + ADP (hexokinase)  G = -8 kcal mol Fructose-6-phosphate + ATP  fructose-1,6-biphosphate + ADP (phosphofructokinase)  G = -5.3 kcal mol Phosphoenolpyruvate + ADP  pyruvate + ATP (pyruvate kinase)  G = -4 kcal mol -1 These three reactions must be bypassed in gluconeogenesis In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose. However, gluconeogenesis is not a reversal of glycolysis. There are three irreversible reactions in glycolysis catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.

1. Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Pyruvate + CO 2 + ATP + H 2 O  oxaloacetate + ADP + P i + 2H + Oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO 2 2. Fructose 6-phosphate is formed from fructose 1,6- bisphosphate. Enzyme - fructose 1,6-bisphosphatase. Fructose 1,6-bisphosphate + H 2 O  fructose 6-phosphate + P i 3. Glucose is formed by hydrolysis of glucose 6-phosphate in a reaction catalyzed by glucose 6-phosphatase. Glucose 6-phosphate + H 2 O  glucose + P i Bypassed Reactions in Gluconeogenesis

Gluconeogenesis The distinctive reactions are shown in red.

Comparison of glycolysis and gluconeogenesis

Bypass I: Pyruvate  Phosphoenolpyruvate Enzyme pyruvate carboxylase is present only in mitochondria. Pyruvate is transported into mitochondria from cytoplasm; the part of pyruvate is formed in mitochondria from amino acids. Essential cofactor of pyruvate carboxylase is biotin, which serves as a carrier of CO 2. The first step in gluconeogenesis is the carboxylation of pyruvate to form oxaloacetate at the expense of a molecule of ATP. Biotin-binding domain of pyruvate carboxylase Structure of carboxybiotin

Oxaloacetate is polar molecule and can not pass through the mitochondria membrane into cytoplasm Therefore it is reduced: oxaloacetate + NADH 2  malate + NAD + Enzyme – malate dehydrogenase Malate passes through the mitochondria membrane into cytoplasm and again oxidized to oxaloacetate (enzyme malate dehydrogenase): malate + NAD +  oxaloacetate + NADH 2 Cytoplasmic oxaloacetate is decarboxylated to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase

Reaction takes place in the cytosol. In decarboxylation reaction GTP donates a phosphoryl group. Oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase. One molecule of ATP and one molecule of GTP were spent to lift pyruvate to the energy level of phosphoenlpyruvate. Phosphoenolpyruvate carboxykinase reaction

Mechanism of phosphoenolpyruvate carboxykinase reaction

A metabolically irreversible reaction The enzyme responsible for this step is fructose 1,6-bisphosphatase F1,6BPase is allosterically inhibited by AMP and fructose 2,6-bisphosphate (F2,6BP) Bypass II: Fructose 1,6-bisphosphate  Fructose 6-phosphate

Bypass III: Glucose 6-phosphate  glucose In most tissues, gluconeogenesis ends with the formation of glucose 6-phosphate (G-6P). Glucose 6-phosphate, unlike free glucose, cannot diffuse out of the cell. The generation of free glucose is controlled in two ways:  enzyme responsible for the conversion of glucose 6-phos- phate into glucose, glucose 6-phosphatase, is regulated;  enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis — liver and to a lesser extent kidney, pancreas, small intestine.

Accumulation of acetyl CoA from fatty acid oxidation signals abundant energy, and directs pyruvate to oxaloacetate for gluconeogenesis. Pyruvate carboxylase reaction Pyruvate carboxylase is allosterically activated by acetyl CoA. biotin This reaction takes place in mitochondria matrix.

The final step in the generation of glucose does not take place in the cytosol. G-6-P is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed by glucose 6- phosphatase, which is bound to the membrane. Glucose 6-phosphatase reaction

Generation of glucose from glucose 6-phosphate. Several proteins play a role in the generation of glucose. T1 transports G-6-P into the lumen of the ER; T2 and T3 transport Pi and glucose respectively back into the cytosol. SP – Ca- binding protein. Ca 2+ -binding stabilizing protein is essential for phosphatase activity. Glucose and P i are then shuttled back to the cytosol by transporters.

2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H 2 O  Glucose + 2 NAD ADP + 2 GDP + 6 P i + 2 H + The Net Reaction of Gluconeogenesis  G°' = -9 kcal mol -1 Six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the conversion of glucose into pyruvate. The extra cost of gluconeogenesis is four high phosphoryl-transfer potential molecules per molecule of glucose synthesized from pyruvate.

Gluconeogenesis enzymes are cytosolic except: (1) Glucose 6-phosphatase (endoplasmic reticulum) (2) Pyruvate carboxylase (mitochondria) (3) Phosphoenolpyruvate carboxykinase (cytosol and/or mitochondria) Subcellular Locations of Gluconeogenic Enzymes

The amounts and activities of the distinctive enzymes of each pathway are controlled. The rate of glycolysis is determined by the concentration of glucose. The rate of gluconeogenesis is determined by the concentrations of precursors of glucose. Regulation of Gluconeogenesis Gluconeogenesis and glycolysis are reciprocally regulated - within a cell one pathway is relatively inactive while the other is highly active.

AMP stimulates phospho- fructokinase, whereas ATP and citrate inhibit it. Fructose 1,6- bisphosphatase is inhibited by AMP and activated by citrate. Fructose 2,6-bisphosphate strongly stimulates phospho- fructokinase 1 and inhibits fructose 1,6-bisphosphatase. During starvation, gluconeo- genesis predominates because the level of F-2,6-BP is very low. High levels of ATP and alanine, which signal that the energy charge is high and that building blocks are abundant, inhibit the pyruvate kinase. Pyruvate carboxylase is activated by acetyl CoA and inhibited by ADP. ADP inhibits phosphoenol-pyruvate carboxykinase. Gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.

Hormones affect gene expression primarily by changing the rate of transcription. Insulin, which rises subsequent to eating, stimulates the expression of phosphofructokinase and pyruvate kinase. Glucagon, which rises during starvation, inhibits the expression of these enzymes and stimulates the production of phosphoenolpyruvate carboxykinase and fructose 1,6- bisphosphatase. Transcriptional control in eukaryotes is much slower than allosteric control; it takes hours or days in contrast with seconds to minutes. Regulation of the Enzymes Amount by Hormones

Any metabolite that can be converted to pyruvate or oxaloacetate can be a glucose precursor Major gluconeogenic precursors in mammals: (1) Lactate (2) Most amino acids (especially alanine), (3) Glycerol (from triacylglycerol hydrolysis) Precursors for Gluconeogenesis