10.1. Conversion of pyruvate to PEP
The first step in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvic acid (PEP). In order to convert pyruvate to PEP there are several steps and several enzymes required. Pyruvate carboxylase, PEP carboxykinase and malate dehydrogenase are the three enzymes responsible for this conversion. Pyruvate carboxylase is found on the mitochondria and converts pyruvate into oxaloacetate. Because oxaloacetate cannot pass through the mitochondria membranes it must be first converted into malate by malate dehydrogenase. Malate can then cross the mitochondria membrane into the cytoplasm where it is then converted back into oxaloacetate with another malate dehydrogenase. Lastly, oxaloacetate is converted into PEP via PEP carboxykinase. The next several steps are exactly the same as glycolysis. Only the process is in reverse to glycolysis.
Pyruvate Carboxylase requires biotin, a cofactor that is covalently attached to a lysine residue. Biotin is a carrier of activated carbon dioxide, just as acyl-CoA carries activated acyl groups.
10.2. Regulation of pyruvate carboxylase
Pyruvate carboxylase is allosterically activated by acetyl-CoA. When the concentration of ATP is low and/or the concentration of acetyl-CoA’s is low, then pyruvate is directed into the citric acid cycle to promote the synthesis of ATP. High concentrations of ATP and acetyl-CoA’s are signals that the cells energy level is high and metabolites are converted into glucose. If the energy status of the cell is low, then the concentrations of ATP and acetyl-CoAs are also low and thus pyruvate is directed towards the TCA cycle for production of more ATP.
10.3. PEP Carboxykinase
PEP carboxykinase simultaneously decarboxylates and phosphorylates oxaloacetate and form PEP. The decarboxylation reaction is very exergonic and helps to drive the otherwise highly endergonic reaction of PEP synthesis.
10.4. Compartmentalization of pyruvate carboxylase and PEP carboxykinase
Pyruvate carboxylase is found only in the matrix of the mitochondra. The next enzyme involved in gluconeogenesis is PEP carboxykinase is found in both the mitochondria or in the cytosol or both depending on the organism and the tissue. In human livers, PEP carboxykinase is found in both the mitrochondria and the cytosol. Pyruvate is transported into the mitochondria where it is either converted into acetyl-CoA by pyruvate dehydrogenase and enters the citric acid cycle, or it is converted into oxaloacetate by pyruvate carboxylase and used for gluconeogenesis.
In tissues where PEP carboxykinase is found only in the mitochondria, oxaloacetate is converted into PEP in the mitochondria. PEP can be transported to the cytosol. In tissues where PEP carboxykinase is found only in the cytosol, oxaloacetate must be reduced to malate so that it can be transported across the mitochondrial membrane to the cytosol where the malate is reoxidized into oxaloacetate and goes on through gluconeogenesis. In humans this enzymatic activity is found in both compartments.
Because the inner mitochondrial membrane is impermeable to OAA, cells that lack mitochondrial PEP carboxykinase transfer OAA into the cytoplasm by using the malate shuttle. In this process, OAA is converted into malate by mitochondrial malate dehydrogenase. After the transport of malate across mitochondrial membrane, the reverse reaction (to form OAA and NADH) is catalyzed by cytoplasmic malate dehydrogenase. The malate shuttle allows gluconeogenesis to continue because it provides the NADH required for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase.
After PEP is formed by PEP carboxykinase, PEP is hydrated by enolase to form 2-phosphoglycerate which in turn is isomerized into 3-phosphoglycerate by phosphoglycerate mutase, phosphoglycerokinase uses ATP to produce 1, 3-bisphosphoglycerate which is reduced by glyceraldehydes-3-phosphate dehydrogenase (GAPDH) into glyceraldehydes-3-phosphate. Triose phosphate isomerase produces dihydroxyacetone phosphate. Aldolase catalyzes an aldol condensation of DHAP and glyceraldehyde-3P to form fructose-1, 6-bisphosphate. All of these enzymes function in both glycolysis and gluconeogenesis because they are all near equilibrium and reversible.
10.5. Conversion of fructose-1, 6-bisphosphate to fructose-6-phosphate
The phosphorylation of fructose-6-phosphate by phosphofructokinase is the first committed step of glycolysis and is irreversible. For gluconeogenesis, fructose-1, 6-bisphosphate is hydrolyzed by the enzyme fructose-1,6-bisphosphatase, an allosterically regulated enzyme. The standard change in free energy for this reaction is ΔGo’ = −16.7 kJ/mol.
The irreversible PFK-1–catalyzed reaction in glycolysis is bypassed by fructose-1, 6-bisphosphatase enzyme. This enzyme is present in cytoplasm of liver and kidney cell. This is an exergonic and irreversible reaction.
This exergonic reaction (G –16.7 kJ/mol) is also irreversible under cellular conditions. ATP is not regenerated, and inorganic phosphate (Pi) is released in cytoplasm. Fructose-1,6-bisphosphatase is an allosteric enzyme. Like other allosteric enzyme, its activity is stimulated by citrate and inhibited by AMP and fructose-2, 6-bisphosphate.
10.6. Formation of glucose from glucose-6-phosphate
Glucose-6-phosphatase enzyme, found only in the endoplasmic reticulum of liver and kidney, catalyzes the irreversible hydrolysis of glucose-6-phosphate to form glucose and Pi. Subsequently this glucose is released into the blood and taken by cells.
Each set of such paired reactions is referred to as a substrate cycle. Because they are coordinately regulated (an activator of the enzyme catalyzing the forward reaction serves as an inhibitor of the enzyme catalyzing the reverse reaction), very little energy is wasted, even though both enzymes may be operating at some level at the same time.
Flux control (regulation of the flow of substrate and removal of product) is more effective if transient accumulation of product is funneled back through the cycle. The catalytic velocity of the forward enzyme will remain high if the concentration of the substrate is maximized. The gain is more in catalytic efficiency than the small energy loss in recycling the product.
10.7. Energy Balance :
Gluconeogenesis is an energy-consuming process. Instead of generating ATP (as in glycolysis), gluconeogenesis requires the hydrolysis of six high energy phosphate bonds.
Patients with von Gierke’s disease lack glucose-6-phosphatase activity in the liver. This resulted into accumulation of excess glucose in the form of Glycogen. Upon fasting the patients are not able to convert this glycogen into glucose. Thus fasting hypoglycemia and lactic acidosis are two prominent symptoms of this disorder.
10.8. Gluconeogenesis Substrates
Lactate is released by red blood cells and other cells that lack mitochondria or during low oxygen concentrations. During exercise lactate is released by skeletal muscle. This lactate is converted into pyruvate and subsequently into glucose by gluconeogenesis. This cycle is known as Cori cycle.
In adipose tissue the store fat is in the form of triacylglycerol. TAG yields glycerol and fatty acid upon metabolism. Glycerol is transported to the liver and then converted to glycerol-3-phosphate by glycerol kinase. Oxidation of glycerol-3-phosphate to form DHAP occurs when cytoplasm NAD+ concentration is relatively high.
10.9. The Glucose-Alanine Cycle
Alanine is formed from pyruvate in muscle. During starvation, it is transported to the liver, alanine is reconverted to pyruvate by alanine transaminase in liver. Eventually pyruvate is used in the synthesis of new glucose by gluconeogenesis process. The glucose-alanine cycle is used to transfer amino nitrogen to the liver. Of all the amino acids that can be converted to glycolytic intermediates (molecules referred to as glucogenic), alanine is the most important amino acid. When exercising muscle produces large quantities of pyruvate, some of these molecules are converted to alanine by a transamination reaction involving glutamate. After it has been transported to the liver, alanine is reconverted to pyruvate and then to glucose.
The glucose-alanine cycle serves several purposes.
In the recycling of the alpha Keto acids between muscle and liver.
It is the mechanism for transporting amino nitrogen to the liver. Because muscle cannot synthesize urea from amino nitrogen, so the amino nitrogen is transported to liver for urea synthesis. Once alanine reaches the liver, it is reconverted to pyruvate. The amino nitrogen of alanine is then incorporated into urea or transferred to other a-keto acids to restore the amino acid balance in the liver.
10.10. Gluconeogenesis Regulation
Like any other metabolic pathways, the rate of gluconeogenesis is regulated. Three parameters that affects the rate of gluconeogenesis are substrate availability, allosteric effector, and hormones.
- Gluconeogenesis is stimulated by high concentrations of lactate, glycerol, and amino acids.
- A high-fat diet, starvation, and prolonged fasting make large quantities of these molecules available.
- The four key enzymes in gluconeogenesis (pyruvate carboxylase, PEP carboxykinase, fructose-1, 6-bisphosphatase, and glucose-6-phosphatase) are affected to varying degrees by allosteric modulators.
- Fructose-1,6-bisphosphatase is activated by citrate and inhibited by AMP and fructose-2,6-bisphosphate.
- Pyruvate carboxylase is activated by Acetyl-CoA ( produce during Fatty acid oxidation and Acetyl-CoA concentration is high during starvation).
4. Hormonal Regulation
- Glucagon hormone decrease the synthesis of fructose-2,6-bisphosphate. Fructose- 2,6-bisphosphate is positive allosteric regulator of PFK1. So PFK 1 is inhibited. Inhibition results into less synthesis of fructose-1,6-bisphosphatase. And eventually less synthesis of PEP. Hence PEP is no longer available for glycolytic enzyme pyruvate kinase. So the activity of pyruvate kinase is inhibited.
- Cortisol hormone increase the expression of gluconeogenic enzymes.
- Insulin hormone increase the synthesis of new molecules of glucokinase, PFK-1 (SREBP1c- induced) and PFK-2 (glycolysis favored). Insulin Hormone also decrease the synthesis of of PEP carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase.
- Glucagon action leads to the synthesis of additional molecules of PEP carboxykinase, fructose-1, 6-bisphosphatase, and glucose-6-phosphatase.
10.11. Catabolism of sugars other than glucose
Starch is the most abundant carbohydrate in our diet. Lactose and sucrose are disaccharides.
Fructose degradation, also called fructolysis, runs mostly in the liver.
- Fructokinase phosphorylates fructose and converted into fructose-1-phosphate.
- Aldolase cleaves it into dihydroxyacetone phosphate and and glyceraldehydes. dihydroxyacetone phosphate enter into glycolysis. Glyceraldehydes also enter into glycolysis after phosphorylation by glyceraldehyde kinase.
- Glyceraldehyde can alternatively be utilized by conversion to glycerol and then to glycerol-1-phosphate. glycerol-1-phosphate is involved in synthesis of triacylglycerol (Fat).
Fructose and sucrose promote obesity more strongly than equivalent amounts of starch or glucose this is because of the above reason as the intermediate is involved in fat synthesis.
10.12. Fructose intolerance
Fructose intolerance is due to mutation in aldolase B gene. In this condition, fructose is still phosphorylated by fructokinase. The product fructose-1-phosphate, is not processed further, and therefore the phosphate tied up in it cant be reused. Since phosphate is required for the regeneration of ATP from ADP, this means that ATP will be lacking too, which will sooner or later damage or even destroy the cell. The disease is characterized by potentially severe liver failure.
Fructosuria is increased in fructose concentration in Urine. This is due to defect in the gene encoding fructokinase. In this disease fructose levels are increased both in the blood and the urine. Since fructose is not phosphorylated, no phosphate depletion occurs, and the liver cells do not occur any damage. The disease is therefore quite benign.
10.13. The Leloir pathway for galactose utilization
This is a galactose utilization cycle mainly occur in liver.
- Galactokinase transfer the phosphate group to galactose and convert galactose into galactose-1-phosphate.
- Galactose-1-phosphate uridyltransferase catalyses the exchange reaction in which the galactose-1-phosphate undergoes an exchange reaction with UDP-glucose and releases glucose-1-phosphate and UDP-galactose.
10.14. Lactose intolerance
It is due to deficiency of lactase enzyme in the small intestine. Lactose is cleaved by lactase enzyme in small intestine. If lactose is not cleaved, it cannot be absorbed by brush border epithelial of small instestine. It enters into large intestine where lactose is metabolised by intestinal bacteria like E.coli. Escherichia coli has a pathway called mixed acid fermentation which produce acids( formic acid) and gas. Formic acid lyases converts formic acid into H2 and CO2. Treatment consists in emission of lactose in the diet.
10.15. Galactosemia means “galactose in the blood”
Galactosemia is due to defect in the following enzymes
Sorbitol is not a sugar, since it lacks a keto or aldehyde group.
It is normally a minor component of dietary carbohydrates. Sorbitol is also formed by metabolism from glucose in the polyol pathway. Sorbitol converted into fructose. NADPH is used in the first step and NAD+ is used in the second.
Aldose reductase can reduce galactose into galactitol. Elevated level of galactose in the blood causes galactitol to accumulate in the lens and causes cataract. Conversion of glucose to fructose via the polyol pathway occurs in the seminal Vesicles in male. Fructose is found in the sperm fluid. Sperm cells require fructose to sustain their motility
Mannose is the C2 epimer of glucose. Mannose is a substrate for hexokinase which converts it into Mannose 6-phosphate. An enzyme similar to phosphoglucose isomerase, phosphomannose isomerase isomerizes mannose 6-phosphate into fructose-6-phosphate. Fructose 6-phosphate is the substrate for PFK-1.