PARV JAIN Class – 10th

PARV JAIN Class – 10th.

Gluconeogenesis: an intro • Defined as biosynthesis of glucose from non-carbohydrate precursors. The major non-carbohydrate precursors are lactate, amino acids, glycerol and the carbon skeletons of most amino acids • Non-carbohydrate precursors of glucose are first converted into pyruvate or as oxaloacetate and DHAP • When fasting, most of the body's glucose needs must be met by gluconeogenesis • Occurs mainly in liver and to some extent in kidney • Responsible for 64% of total glucose production over the first 22 hours of the fast and accounts for almost all the glucose production by 46 hours.

Glucose glucose 6— phosphatase Glucose—6—P Fructose—6—P fructose bisphosphatase Fructose— 1 , 6—P The pathway 1,3-Bisphosphoglycerate ATP 3-Phosphoglycerate NADH+ H+ NAD+ Glyceraldehyde 3- P DHAP Glycerol Glycerol 3- P 2-Phosphoglycerate Phosphoenolpyruvate GDP : carboxykinase GTP OAA Pyruvate Lactate pyruvate : carboxylase Alanine, O Amino acids TCA cycle Amino acids.

Thermodynamic Barriers • Gluconeogenesis is not reversal of glycolysis. • There are three major thermodynamic barriers for the pathway which are three irreversible steps in glycolysis • These three major barriers are bypassed by successive smaller steps with relatively lesser AG. Glucose + ATP Glucose 6- P + ADP AG = -8.0 kcal/ mol (-33 kJ /mol) Fructose 6 - P+ ATP Fructose 1, 6- P + ADP AG = -5.3 kcal/ mol (-22 kJ /mol) Phosphoenolpyruvate+ ADP Pyruvate + ATP AG = -4.0 kcal/ mol (-17 kJ /mol).

Pyruvate to Phosphoenolpyruvate Endergonic & requires free energy input. • This is accomplished by first converting the pyruvate to oxaloacetate, a "high-energy" intermediate Exergonic decarboxylation OAA provides the free energy necessary for PEP synthesis. • C02 is added to pyruvate by pyruvate carboxylase enzyme • C02 that was added to pyruvate to form OAA is released in the reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK) to form PEP • GTP provides a source of energy & phosphate group of o PEP. o v ate pyruvate carboxylase 1 + ATP ADP + Pi o II II Oxaloacetate PEPCK 2 GTP GDP + C02 Phosphoenol- pyruvate (PEP).

Pyruvate to PEP PYRUVATE CARBOXYLASE tetrameric protein of identical 130-kD subunits •Has a biotin prosthetic group. •Biotin functions as a C02 carrier by acquiring a carboxyl substituent at its ureido group •Biotin is covalently bound to the enzyme by an amide linkage of Lys residue to form a biocytin o o o O o —P—O— Rib ATP HN NH s Adenine o Bicarlx»nate ADP + Pi O -oz NH s Biotinyl-enzyme o O o C—C—CH -O O O c 2 Carboxybiotinyl-enzyme O -o Pyruvate enolate o o Oxaloacetate o HN NH s o Biotinyl-enzyme.

Pyruvate to PEP Oxaloacetate PHOSPHOENOLPYRUVATE CARBOXYKINASE o o •OAA is converted to PEP by PEPCK. •Mg2+-dependent reaction requires GTP as the phosphoryl group donor •Reaction is reversible under intracellular conditions C—CH2 —CA -o o o O o GTP GDP PEP carboxykinase C02 Phosphoenolpyruvate.

Pyruvate to PEP (overall reaction) Pyruvate +ATP + GTP +HCO PEP + GDP +Pi + • AG'O= 0.9 kJ/mol (Vs -17 kJ/mol of glycolysis) for this reaction which make the reaction quite reversible • But actually the AG under cellular condition is strongly negative due to very lesser concentration of PEP favoring a forward way of reaction (-25 kJ/mol) Thus the reaction is strongly irreversible.

Fructose 1,6-Bis P to Fructose 6-P • This step is irreversible hydrolysis of fructose 1,6- bisphosphate to fructose 6-phosphate and Pi. • Fructose 1,6-bisphosphatase (FBPase-1) Mg 2+ dependent enzyme catalyzes this exergonic hydrolysis. • It is present in liver, kidney, and skeletal muscle, but is probably absent from heart and smooth muscle. • it is an allosteric enzyme that participates in the regulation of gluconeogenesis. • A G =- 16.3 kJ/mol (Vs -22 kJ/mol of glycolysis) Fructose 1,6-bisphosphate +1-120 Fructose 6-phosphate + Pi.

Glucose 6-P to Glucose This final step in the generation of glucose does not take place in the cytosol. Glucose 6-P is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6- phosphatase, which is bound to the membrane at the luminal side. This compartmentalisation can only be seen in glucose producing cells like hepatocytes, renal cells and epithelial cells of small intestine An associated Ca2+ binding stabilizing protein is essential for phosphatase activity. Glucose and Pi are then shuttled back to the cytosol by a pair of transporters. Glucose 6-phosphate +1-120 Glucose+ Pi.

Glucose 6-P to Glucose Glucose 6- phosphatase H20 + glucose 6-phosphate T3 Pi + glucose Cytosolic side ER lumen Tl- transports glucose 6-phosphate into the lumen of the ER T 2- transport Pi to the cytosol T 3 — transport glucose to the cytosol. SP- Ca2+binding protein The glucose transporter in the endoplasmic reticulum membrane is like those found in the plasma membrane..

Energetics of gluconeogenesis • 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. Thus it is not a simple reversal of glycolysis but it is energetically an expensive affair. Glycolysis: Glucose + 2NAD+ + 2ADP + 2Pi —+ 2 pyruvate + 2NADH + 41--1 + + 2ATP + 21--120 Gluconeogenesis: 2 Pyruvate + 2NADH + 4H+ + 4ATP + 2GTP + 61120 glucose + 2NAD+ + 4ADP + 2GDP + 6Pi Overall: 2ATP + 2GTP + 41120 2ADP + 2GDP + 4Pi.

Substrates of gluconeogenesis • The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate. Entry of glucogenic amino acids • Amino acids that are degraded to pyruvate, a-ketoglutarate, succinyl CoA, fumarate, or oxaloacetate are termed glucogenic amino acids. • The net synthesis of glucose from these amino acids is feasible because these citric acid cycle intermediates and pyruvate can be converted into phosphoenolpyruvate. • Amino acids are derived from the dietary proteins, tissue proteins or from the breakdown of skeletal muscle proteins during starvation. • After transamination or deamination, glucogenic amino acids yield either pyruvate or intermediates of the citric acid cycle CH3 1 coo- Alanine alanine aminotransferase CH3 coo- Pyruvate.

Regulation of gluconeogenesis • Need of regulation ATP + fructose 6-phosphate PFK-I ADP + fructose 1,6-bisphosphate Fructose 1,6-bisphosphate + H20 FBPase-1 fructose 6-phosphate + Pi The sum of these two reactions is ATP + H20 ADP + Pi + heat • There are three major types of regulation — Allosteric regulation — Hormonal Regulation — Transcriptional Regulation.

Allosteric regulation Phosphofructokinase-I (PFK-I) — Enzyme has several regulatory sites at which allosteric activators or inhibitors bind — ATP inhibits PFK-I by binding to an allosteric site and lowering the affinity of the enzyme for fructose 6-phosphate — ADP and AMP act allosterically to relieve this inhibition by ATP. High citrate concentration increases the inhibitory effect of ATP. — Thus glycolysis is down regulated when enough ATP is present in cells. ATP AMP, ADP Fructose 6- + ATP phosphate citrate fructose 2,6- bisphosphate Fructose 1,6- + ADP bisphosphate.

Hormonal Regulation Stimulates glycolysis, inhibits gluconeogenesis H20 V[F26BP] Inhibits glycolysis, stimulates gluconeogenesis PFK-2 (active) OH FBPase-2 (inactive) phospho- cAMP-dependent protein protein kinase phosphatase ATP glucagon (t [cAMPJ) ADP PFK-2 (inactive) (active) o —P—O- o-.