GLUCONEOGENESIS Summary of handout: Ferchmin 2012/gluconeogenesis GLUCONEOGENESIS Summary of handout: Comparison with glycolysis, unique and shared enzymes Role of biotin in gluconeogenesis (and comparison with vitamin K which is not involved in gluconeogenesis) "Reversal" of pyruvate kinase. Participation of the mitochondria "Reversal" of Phosphofructokinase "Reversal" of hexokinase The Cori and alanine cycles Regulation. Role of insulin and glucagon in glycolysis and gluconeogenesis. Glycogenic and ketogenic compounds Metabolic role of gluconeogenesis

COMPARISON BETWEEN GLYCOLYSIS AND GLUCONEOGENESIS The overall reaction of gluconeogenesis is: COOH | 2 CO + 4 ATP + 2 GTP + 2 NADH + 2H+ + 2 H2O ➔ glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ CH3 ΔG°'= -9 Kcal/mole The overall reaction of glycolysis is: COOH | Glucose + 2 ADP + 2 Pi + 2 NAD+ ➔ 2 CO + 2 ATP + 2 NADH + 2H+ + 2 H2O CH3 ΔG°'= -20 Kcal/mole Glycolysis yield 2 ATP/glucose plus a net dissipated - 9 Kcal/mole. Gluconeogenesis is really bad news, it consumes the equivalent of 6 ATP/glucose synthesized. Why would be a need for such a metabolic pathway?

Gluconeogenesis is the synthesis of glucose from precursors that are not sugars, like lactate, pyruvate, glycerol or glycogenic amino acids. The synthesis of glucose from other sugars simply is not gluconeogenesis. The neo means de novo from non-carbohydrate molecules. There is no gluconeogenesis from fatty acids except the rare ones with odd number of carbons that have a minute contribution to the synthesis of glucose of rather academic value just to stimulate your intellect! Fatty acids contribute to the fasting organism with ATP through β-oxidation and oxidation of ketone bodies in the Krebs cycle. Ketone bodies only partially substitute for glucose and are synthesized by a pathway different from gluconeogenesis. Ketone bodies are potentially dangerous in the absence of glucose (you will study this later). In conclusion: lipids can spare glucose because they provide for ATP that otherwise would be synthesized in glycolysis. But lipids do not substitute glucose. We need about l60 grams of glucose per day, 120 grams are needed for the brain and 40 grams for muscle, erythrocytes, eye lens cells, kidneys medulla, etc. Approximately 200 grams are stored in hepatic glycogen. Gluconeogenesis provides the necessary glucose during fast. The complete gluconeogenesis occurs in liver and a small fraction in kidney. Since glycolysis is irreversible gluconeogenesis cannot be the reversal of glycolysis. The enzymes that catalyze the irreversible reactions in glycolysis are overridden in various ingenious ways in gluconeogenesis.

We will study gluconeogenesis by comparing it with glycolysis How do your reverse an irreversible metabolic step? By using an enzyme that catalyzes the opposite also irreversible step!!! By using an enzyme that catalyzes the opposite also irreversible step!!! How do your reverse an irreversible metabolic step?

The last glycolytic step catalyzed by pyruvate kinase is irreversible, the free energy change is high, -7.5 Kcal/mole. To reverse this step in gluconeogenesis two enzymes are used and the process takes place in two cellular compartments.

__________________________________________________________________ Enzymatic differences between glycolysis and gluconeogenesis a) Regulatory enzymes __________________________________________________________________Glycolysis Gluconeogenesis __________________________________________________________________ Hexokinase Glucose 6-phosphatase Phosphofructokinase Fructose 1,6-bisphosphatase Pyruvate Pyruvate kinase carboxylase Phosphoenolpyruvate carboxykinase __________________________________________________________________b) The remaining enzymes are shared by both pathways __________________________________________________________________Essential concept: Pathways for breakdown and synthesis of a particular metabolite are always different, utilizing unique enzymes in one or more steps. The difference usually is in the regulatory enzymes. Pyruvate carboxylase is mitochondrial and hepatic

The above exergonic reaction is overcome by an input of energy and First we will consider the reversal in gluconeogenesis of the exergonic glycolytic reaction catalyzed by pyruvate kinase. The reaction is shown below: The above exergonic reaction is overcome by an input of energy and of two complex reactions that regenerate phosphoenolpyruvate. The two enzymes involved are: a) Pyruvate carboxylase b) Phosphoenolpyruvate carboxykinase

With reference to pyruvate carboxylase This part is a “pop up” about a different subject marginally related to gluconeogenesis but of practical importance (NBE). There are two vitamins involved in CO2 incorporation in mammalian tissues: biotin and vitamin K. In gluconeogenesis biotin is a cofactor for pyruvate carboxylase. Biotin binds avidly to AVIDIN. Vitamin K is involved in the posttranslational synthesis of γ-carboxyglutamate involved prominently in blood coagulation. Biotin is attached to the amino of lysine in carbon ε. Biotin is a cofactor for 1) Pyruvate carboxylase 2) β-methylcrotonylCoA carboxylase 3) Propionyl CoA carboxylase 4) Acetyl CoA carboxylase Biotin Vitamin K

PYRUVATE CARBOXYLASE is exclusively hepatic. So, after leaving the detour of biotin we return to pyruvate carboxylase and gluconeogenesis PYRUVATE CARBOXYLASE is exclusively hepatic. The reaction catalyzed by pyruvate carboxylase takes place in 2 steps: STEP 1: Enz-Biotin + ATP + CO2 ➔ Enz-Carboxybiotin + ADP + Pi This first step requires CH3-CO-CoA (acetyl~S-CoA) STEP 2: Enz-Carboxybiotin + pyruvate ➔ Enz-Biotin + oxaloacetate This is an anaplerotic reaction (re-supplying). It provides oxaloacetate for the Krebs cycle and for gluconeogenesis. The requirement for CH3-CO~S-CoA is a manifestation of the need of oxaloacetate for the TCA cycle or the abundance of CH3-CO-CoA produced by a lipid rich diet that calls for storage of glucogenic intermediaries.

The next step is the synthesis of phosphoenolpyruvic acid from oxaloacetate The synthesis of PEPA reverses the effect of pyruvate kinase

Gluconeogenesis takes place in the cytosol and in the mitochondria Gluconeogenesis takes place in the cytosol and in the mitochondria. There are two pathways to generate PEPA (phosphoenolpyruvic acid). In both pathways NADH must be generated to allow the activity of glyceraldehyde-3-phosphate dehydrogenase in the reduction of 3-phosphoglyceric acid. From PEPA to fructose-1,6-bisphosphate all the steps are shared by glycolysis and gluconeogenesis and are reversible.

This graph represents the relationship between the activity of both enzymes and the energy status of a muscle cell.

From previous page, we can see the relationship between phosphofructokinase and fructose-1,6-phosphatase In this point we have a metabolic cycle or futile cycle that “wastes” energy but provides more leverage for regulation

Integration of gluconeogenesis and glycolysis

There is a fundamental difference between the role of glycolysis in the “peripheral” organs and the liver. In liver the role of glycolysis is to make you FAT!!!! In muscle is to make you run!!! Ethanol and fatty acids are not glucogenic (odd number fatty acids contribute insignificantly to gluconeogenesis). Glycerol, the ketoacids of most amino acids, lactate and pyruvate ARE glucogenic. Galactose, fructose, etc are not glucogenic. They are monosaccharides in equilibrium with glucose! Warning: whoever says that fatty acids with even number of carbons are glucogenic will be decapitated!

Warning: whoever says that fatty acids with even number of carbons are glucogenic will be decapitated!