Gluconeogenesis is the process of synthesizing glucose from non carbohydrate sources. Gluconeogenesis occurs mainly in liver. Gluconeogenesis occurs to a more limited extent in the kidney and small intestine under some conditions. Definition of Gluconeogenesis: The biosynthesis of a carbohydrate from simpler, non-carbohydrate precursors such as Oxaloacetate and Pyruvate is called “Gluconeogenesis“. Few basics before continue, The starting point of gluconeogenesis is pyruvic acid, although Oxaloacetic acid and dihydroxyacetone phosphate also provide entry points. Lactic acid, some amino acids from protein and glycerol from fat can be converted into glucose. Gluconeogenesis is similar but not the exact reverse of glycolysis, some of the steps are the identical in reverse direction and three of them are new ones. Without going into detail, the general gluconeogenesis sequence is given in the graphic on the left. Notice that oxaloacetic acid is synthesized from pyruvic acid in the first step. Oxaloacetic acid is also the first compound to react with acetyl CoA in the citric acid cycle. The concentration of acetyl CoA and ATP determines the fate of oxaloacetic acid. If the concentration of acetyl CoA is low and concentration of ATP is high then gluconeogenesis proceeds. Also notice that ATP is required for a biosynthesis sequence of gluconeogenesis. Gluconeogenesis occurs mainly in the liver with a small amount also occurring in the cortex of the kidney. Very little gluconeogenesis occurs in the brain, skeletal muscles, heart muscles or other body tissue. In fact, these organs have a high demand for glucose. Therefore, gluconeogenesis is constantly occurring in the liver to maintain the glucose level in the blood to meet these demands. Steps involved in Gluconeogenesis: Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis. Kreb’s pointed out that energy barriers obstruct a simple Reversal of Glycolysis: Between Pyruvate and PEP (Enzymes: Pyruvate Carboxylase and Phosphoenol pyruvate Carboxylase-PEPCK) Between Fructose-1,6-bis P and Fructose-6-P (Enzymes: Fructose-1,6-BisPhosphatse) Between Glucose-6-P and Glucose (Enzymes: Glucose-6-Phosphatase) Between Glucose-1-P and Glycogen (Enzyme: Glycogen Synthase) Three reactions of Glycolysis have forward direction that they are essentially irreversible (see lecture notes on Glycolysis): Hexokinase (or Glucokinase), Phosphofructokinase, and Pyruvate Kinase. These steps must be bypassed in Gluconeogenesis. Two of the bypass reactions involve simple hydrolysis reactions. Below is the forward reaction catalyzed by each of these Glycolysis enzymes, followed by the bypass reaction catalyzed by the Gluconeogenesis enzyme. Step 1: In Glycolysis, First step is Phosphorylation Glucose + ATP –> Glucose-6-phosphate + ADP Enzyme: Hexokinase or Glucokinase (Glycolysis) In Gluconeogenesis, the first step in Glycolysis is reversible. Glucose-6-phosphate + H2O –> glucose + Pi Enzyme: Glucose-6-phosphatase Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells. Evidence indicates that the catalytic site is exposed to the ER lumen. Another subunit of the enzyme is postulated to function as a translocase, providing access of substrate to the active site. Step 2: In Glycolysis, Step 3 is Fructose-6-phosphate + ATP –> fructose-1,6-bisphosphate + ADP Enzymes: Phosphofructokinase In Gluconeogenesis, the step 3 in glycolysis is reversible. Fructose-1,6-bisphosphate + H2O –> fructose-6-phosphate + Pi Enzyme: Fructose-1,6-bisphosphatase Step 3: In Glycolysis, the step 9 is Phosphoenolpyruvate + ADP –> pyruvate + ATP Enzyme: Pyruvate Kinase For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP. In Gluconeogenesis, The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following: a) PEP Carboxylase Reaction Pyruvate + HCO3– + ATP –> Oxaloacetate + ADP + Pi Enzyme: Pyruvate Carboxylase (b) PEP Carboxykinase Reaction: Oxaloacetate + GTP –> Phosphoenolpyruvate + GDP + CO2 Enzyme: PEP Carboxykinase Contributing to spontaneity of the two-step pathway are the following: Free energy of cleavage of one ~P bond of ATP is conserved in the carboxylation reaction.Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction (PEP synthesis). Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP. About Biotin Vitamin: Pyruvate Carboxylase utilizes biotin as prosthetic group. Biotin has a 5-carbon side chain whose terminal carboxyl is in an amide linkage to the e-amino group of a lysine of the enzyme. The biotin and lysine side chains together form a long swinging arm that allows the functional group of biotin to swing back and forth between two active sites. Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase. ATP reacts with HCO3– to yieldcarboxyphosphate. The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring system. Overall: Biotin + ATP + HCO3– –> carboxybiotin + ADP + Pi Biotin dependent enzymes in animals Enzyme Role Pyruvate Carboxylase First reaction in pathway that convers 3-carbon precursors to glucose (gluconeogenesis) Acetyl~coA carboxylase Commits acetate units to fatty acid synthesis by forming malonyl~coA Propionyl~coA Carboxylase Converts propionate to succinate, which can then enter citric acid cycle. beta-Methylcrotonyl~coA carboxylase Catabolism of leucine and certain isoprenoid compounds Other Reactions in Gluconeogenesis: 1. Pyruvate Carboxylase: The enzyme converts pyruvate to oxaloacetate, is allosterically activated by acetyl coenzyme A. The adaptive value of this regulation relates to the interconnectness of the pathways shown at right. Acetyl CoA enters Krebs Cycle by condensing with oxaloacetate, whose concentration tends to be limiting for Krebs Cycle. When Gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose (via PEP). Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The resulting increase in [acetyl CoA] activates Pyruvate Carboxylase to synthesize more oxaloacetate. 2. Lactate to Glucose: The major breakdown product of anaerobic glycolysis in muscle is lactic acid. Muscle tissue is calledLactic acid. Muscle tissue is, however, not capable of re-synthesizing glycogen from lactate. This conversion therefore, takes place entirely in liver. Muscle lactate is transported by the blood to liver where it is converted to Glucose and glycogen by enzymes involved in gluconeogenesis. Liver glycogen then breaks down to glucose and is carried back to muscles by blood. This conversion of muscle lactic acid to glucose in liver and its re-entry into muscle is called Cori cycle. 3. Amino acids to Glucose: The major portion of glucose formed in gluconeogenesis come from amino acids. Glycogenic amino acids are converted to either citric acid cycle intermediates or pyruvate. These substances from glucose in liver. Pyruvate is carboxylated to form OAA by pyruvate carboxylase and ATP in mitochondria. Further reactions in the formation of glucose take place in cytoplasm and therefore oxaloacetate must come out of mitochondria. Oxaloacetate, however, does not readily permeate through mitochondrial membrane and thus requires conversion to a compound which could diffuse out of mitochondria. This is achieved mainly by its conversion to malate which readily passes through mitochondrial membrane. In cytoplasm, malate is reconverted to oxaloacetate. Oxaloacetate is then decarboxylated to form phosphoenol pyruvate (PEP) by phosphoenol pyruvate carboxylase and GTP. The conversion of phosphoenol pyruvate (PEP) to fructose-1, 6-diphosphate is carried out by enzymes of glycolysis found in all tissues. The hydrolysis of fructose diphosphate to form fructose-6-phosphate requires a specific fructose diphosphatase. Frusctose-6-phosphate also requires a specific glucose-6-phosphatase for its conversion to Glucose. Both the abovementioned specific enzymes are found only in liver and kidney tissues. 4. Glycerol to Glucose: Glycerol arising from the breakdown of triacylglycerides is also a good source for the synthesis of glucose in liver. It requires initial phosphorylation by ATP followed by reduction to form DHAP (Dihydroxyacetone phosphate) which enters the pathway of gluconeogenesis. Importance of Gluconeogenesis: A continual supply of Glucose is necessary as a source of energy, especially for the Nervous system and the Erythrocytes. Gluconeogenesis mechanism are used to clear the products of the metabolism of other tissues from the blood, eg: Lactate, produced by Muscle and erythrocytes and Glycerol, which is continuously produced by adipose tissue. Source
