GLYCOLYSIS Learning objectives: List the enzymes and intermediates involved in glycolysis List the irreversible and regulated steps of glycolysis Discuss regulation of glycolysis

Glucose transporters NameTissue locationKm. GLUT1 All mammalian tissues1 mM Basal glucose uptake GLUT2 Liver and pancreatic β cells15-20 mMUptake approximately proportional to glucose conc. GLUT3 All mammalian tissues1 mM Basal glucose uptake GLUT4 Skeletal muscle and fat5 mMRecruited to cell membrane in insulin dependent manner Blood glucose concentrations Normal plasma glucose concentrations roughly 3.9 – 8.3 mM

Glucose transporters

GLUT4 is recruited to cell membrane by insulin-dependent translocation The increase in number of GLUT4 transporters in the cell membrane increases the speed of glucose transport. Insulin stimulates glucose uptake in fat tissue and skeletal muscle

Glycolysis Stoichiometry 1 Glucose + 2 ADP + 2 Pi + 2 NAD + → 2 Pyruvate + 2 ATP + 2 H 2 O + 2 NADH + 2 H + (Emden-Meyerhof glycolytic pathway) Aerobic conditions: Oxygen is required to re-oxidize NADH to NAD + via the electron transport chain Anaerobic conditions: NADH is re-oxidized to NAD+ in a reaction in which pyruvate is reduced to lactate (humans) or ethanol (yeast and other microorganisms) Glycolysis is used to provide energy (in the form of ATP) and intermediates for other metabolic pathways. Glycolysis occurs in the cytoplasm.

C 6 H 12 O 6 O O - C C O CH 3 Pyruvate Glucose O O - C HO C H CH 3 Lactate H HO C H CH 3 Ethanol CO 2 + H 2 O + O 2 GLYCOLYSIS FERMENTATION TCA CYCLE & OXIDATIVE PHOSPHORYLATION

O H C H- C - OH CH 2 OH C H 2 OH C O CH 2 OH CH 2 OH H- C - OH CH 2 OH O OH C H- C - OH CH 2 OH Glycerol Glyceraldehyde Glyceric acid Glycerate O O - C H- C - OH CH 2 OH O O - C C O CH 3 Dihydroxyacetone Pyruvate (α-keto carboxylic acid)

Glucokinase Hexokinase R kinase R-OH + ATP R-OPO ADP Irreversible Highly exergonic (ΔG = kJ/mol) Regulated step in glycolysis Glucose + ATP Glucose 6-phosphate + ADP

Normal physiological range Glucokinase is a glucose sensor

Ca Glucose GK G-6-PPyruvate ATP ADP K + Depolarization 2+ Ca 2+ Insulin GLUT2 Glucose-stimulated insulin secretion in pancreatic beta cells Glucose metabolism determines secretion of insulin

GLUT2 is a high Km, high efficiency glucose transporter (not rate-limiting) Glucokinase has a high Km (is rate-limiting) - reaction velocity is roughly proportional to glucose concentration - therefore glucose metabolism and insulin secretion in beta cells is roughly proportional to glucose concentration

Phosphoglucose isomerase Glucose 6-phosphate Fructose 6-phosphate

Irreversible Highly exergonic (ΔG = kJ/mol) Regulated step in glycolysis The main regulatory enzyme of the glycolytic pathway ATP balance: 2 ATP spent in phosphorylating glucose and fructose 6-phosphate Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP Phosphofructokinase

CH 2 OPO 3 2- Ι C = O Ι HO – C – H Ι H – C – OH Ι H – C – OH Ι CH 2 OPO 3 2- Fructose 1,6-bisphosphate CH 2 OPO 3 2- Ι C = O + Ι CH 2 OH Dihydroxyacetone phosphate CHO Ι H – C – OH Ι CH 2 OPO 3 2- Glyceraldehyde 3-phosphate Aldolase

CH 2 OPO 3 2- Ι C = O Ι CH 2 OH Dihydroxyacetone phosphate CHO Ι H – C – OH Ι CH 2 OPO 3 2- Glyceraldehyde 3-phosphate Triose phosphate isomerase

Mixed anhydride of phosphoric acid and a carboxylic acid High energy bond Energy from oxidation of glyceraldehyde 3-phosphate to the carboxylic acid is “captured” in the formation of the acyl~phosphate linkage CHO Ι H – C – OH Ι CH 2 OPO 3 2- Glyceraldehyde 3-phosphate C Ι H – C – OH Ι CH 2 OPO ,3-Bisphosphoglycerate 2- O 3 PO O + NAD + + P i + NADH + H + Glyceraldehyde 3-phosphate dehydrogenase

Substrate-level phosphorylation ATP balance: 2 ATP spent in phosphorylating glucose and fructose 6-phosphate 2 x 1 ATP generated in the phosphoglycerate kinase step 1,3-Bisphosphoglycerate + ADP 3-Phosphoglycerate + ATP Phosphoglycerate kinase

3-Phosphoglycerate 2-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate + H 2 O Phosphoglycerate mutase Enolase

Substrate-level phosphorylation Irreversible Highly exergonic (ΔG = kJ/mol) Regulated step in glycolysis Phosphoenolpyruvate + ADP Pyruvate + ATP Pyruvate kinase

ATP balance: 2 ATP spent in phosphorylating glucose and fructose 6-phosphate 2 x 1 ATP generated in the phosphoglycerate kinase step 2 x 1 ATP generated in the pyruvate kinase step 2 molecules of ATP are generated from 1 molecule of glucose in glycolysis Phosphoenolpyruvate + ADP Pyruvate + ATP Pyruvate kinase

Stoichiometry 1 Glucose + 2 ADP + 2 Pi + 2 NAD + → 2 Pyruvate + 2 ATP + 2 H 2 O + 2 NADH + 2 H + The 3 Irreversible Enzyme-catalyzed Reactions 1.Hexokinase (or glucokinase) 2.Phosphofructokinase 3.Pyruvate kinase

In yeast and many other microorganisms: Pyruvate + H + Acetaldehyde + CO 2 Acetaldehyde + NADH + H + Ethanol + NAD + Pyruvate decarboxylase Alcohol dehydrogenase

In mammals: Under anaerobic conditions, e.g. in rapidly contracting muscle In erythrocytes Under aerobic conditions NADH + H + + ½ O 2 NAD + + H 2 O Electron transport chain Pyruvate + NADH + H + Lactate + NAD + Lactate dehydrogenase

Fates of pyruvate generated by glycolysis Pyruvate Acetyl CoA Oxaloacetate Alanine Oxidation via TCA cycle Fatty acid synthesis Lactate PEP

Regulation of metabolic pathways The flow of intermediates through metabolic pathways is controlled by four mechanisms 1.Substrate availability 2.Allosteric activation or inhibition 3.Covalent modification of enzymes 4.Induction/repression of enzyme synthesis

Signals Energy state ATP high-energy signal ADP, AMP low-energy signal Availability of biosynthetic intermediates Citrate Alanine Metabolic state of the whole organism Insulin – signal of the fed state High blood glucose – signal of the fed state Glucagon – signal of the fasted state Epinephrine – signal of stress Metabolic regulation of biochemical pathways makes “physiological sense” Control of flow though a pathway is often distributed among several enzymatic steps in the pathway

Fed state Fasted state glucose P GK glucose glucose-6-phosphate GLUT 2 Insulin receptorInsulin Lipids Pyruvate Hepatic glucose uptake and utilization Hepatic glucose production glucose P glucose-6-phosphate GLUT 2 Receptor Glucagon Glycogen Gluconeogenesis G6Pase Liver cell - Hepatocyte Glycolysis Glycogen

Rapidly contracting state Resting state glucose P Hexokinase glucose glucose-6-phosphate GLUT 4 glucose P glucose-6-phosphate GLUT 4 Glycogen Skeletal muscle cell Glycogen Low ATP High AMP ATP Glycolysis High ATP Low AMP ATP

Hexokinase Glucose 6-phosphate (low phosphofructokinase activity) Glucokinase High blood glucose (release from GKRP, High K m ) Insulin stimulates gene transcription (only in liver) Glucokinase Hexokinase Glucose + ATP Glucose 6-phosphate + ADP

Glucokinase (GK) can be bound to the Glucokinase Regulatory Protein (GKRP). When GK is bound to the GKRP, it is sequestered in the nucleus and is inactive. Glucose promotes dissociation of GK from the GKRP, allowing GK to be transported to the cytoplasm where it is active in glycolysis.

The main regulatory step of glycolysis ATP (high-energy state) Citrate (abundance of biosynthetic building blocks) H + (build-up of lactate) AMP (low-energy state) Fructose 2,6-bisphosphate (fed state, high insulin/glucagon ratio) - - Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP Phosphofructokinase

ATP is a substrate for phosphofructokinase ATP can also bind to an allosteric site Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP Phosphofructokinase

ATP (high-energy state) Alanine (abundance of alanine which is synthesized from pyruvate and is an important gluconeogenic substrate) Glucagon (fasted state) Fructose 1,6-bisphosphate (feed-forward mechanism) Glucose (stimulates gene transcription) Phosphoenolpyruvate + ADP Pyruvate + ATP Pyruvate kinase

Occurs in liver in the fasted state Glucagon binding to the glucagon receptor  Activation of adenylyl cyclase  Increase in [cAMP]  Activation of protein kinase A  Phosphorylation and inactivation of pyruvate kinase