Glycolysis

Metabolism Definition: The sum total of chemical reactions occurring in cells. Major Divisions: Catabolism: Energy yielding (ATP yielding) conversion of fuels to end-products. Anabolism: Energy requiring (ATP requiring) biosynthetic processes

Glucose Transport (Glucose)out (Glucose)in Glycolysis occurs in the cytosol. Glucose must be transported into the cell from the extracellular fluid: Glucose transporters (GLUT) are involved. Different tissues have different glucose transporters with varying properties. In some tissues, e.g., muscle and fat, this transport requires the hormone, insulin. (Glucose)out (Glucose)in

Overview of Glycolysis

Hexokinase Reaction Glucose + ATP Glucose 6-phosphate + ADP The reaction is irreversible. Hexokinase is an allosteric enzyme inhibited by its product, Glucose 6-P. Hexokinase is found in most cells.

Hexokinase and Glucokinase The liver has a specialized hexokinase, called glucokinase, suited to the liver’s role as chief feeder of other tissues in the body. The kinetic properties of glucokinase are suited to the fact that blood arriving in the liver from the intestines will have widely varying glucose concentrations, depending upon dietary state.

Kinetic Properties of Hexokinase and Glucokinase Most tissues Phosphorylates several hexoses Inhibited by glucose 6-P Low Km & Vmax Glucokinase Hepatocytes & pancreatic islet cells Not inhibited by glucose 6-P High Km & Vmax

Phosphoglucoisomerase Glucose 6-phosphate = Fructose 6-phosphate Reaction is reversible

Phosphofructokinase 1 (PFK-1) Fructose 6-phosphate + ATP Fructose 1,6 Bisphosphate + ADP

Phosphofructokinase 1 (PFK-1) Fructose 6-phosphate + ATP Fructose 1,6 Bisphosphate + ADP Reaction is irreversible Most important control point in the glycolytic pathway ATP inhibits; AMP and fructose 2,6 bisphosphate activate Enzyme has two binding sites for ATP, one a substrate-binding site and the other allosteric

PFK-2 & Fructose 2,6 Bisphosphate

Irreversible Reactions and Regulation Regulation typically occurs at irreversible steps. If a process is reversible, that is, if it is at equilibrium, making an enzyme more active will have no effect because the process has nowhere to go.

PFK-1 Kinetics

Fructose Bisphosphate Aldolase Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate The top half of the hexose bisphosphate becomes DHAP and the bottom half becomes Glyceraldehyde 3-P (G3P)

Triose Phosphate Isomerase This step interconverts the two triose phosphates, G3P and DHAP: G3P = DHAP Since only G3P has a further fate in the glycolytic pathway, it is as if two G3P molecules are produced per glucose molecule.

Properties of NAD+ NAD is Nicotinamide Adenine Dinucleotide NAD+ is an intermediate oxidizing agent in cells. It can accept two hydrogens to be reduced to NADH + H+ XH2 + NAD+ = X + NADH + H+ When the two hydrogens are transferred, one is transferred in the medium as a proton, H+, and the other as a hydride ion, H- , which is attached to the top of the nicotinamide ring.

NAD Structure

Glyceraldehyde Phosphate Dehydrogenase G3P + NAD+ + Pi = 1,3-BPG + NADH + H+ The phosphate at position 1 is a phosphoanhydride and has a high free energy of hydrolysis. The phosphate at position 3 is a phosphoester and has a lower free energy of hydrolysis

Phosphoglycerate Kinase 1,3-BPG + ADP = 3-PGA + ATP The high-energy phosphate at position 1 (the phosphoanhydride) can be transferred to ADP to generate an ATP.

Net Yield of the Last Two Reactions: G3P + NAD+ + ADP = 3-PGA + ATP + NADH + H+ One ATP is synthesized per molecule of G3P oxidized. Since one glucose furnishes two triose phosphates, this process produces a net yield of 2 ATP per glucose.

How 2,3-BPG is made in RBCs Most cells have only trace amounts of 2,3-Bisphosphoglycerate (2,3-BPG) RBC make high levels of 2,3-BPG using this side reaction of glycolysis

Phosphoglycerate Mutase 3-PGA = 2-PGA

Enolase 2-PGA = PEP PEP is phosphoenolpyruvate. It is a high-energy phosphate compound.

Pyruvate Kinase PEP + ADP Pyruvate + ATP Reaction is irreversible and regulatory. Allosteric activator: F1,6BP, an example of feed-forward activation. Allosteric inhibitor: ATP A net of 2 ATP is produced by this step per glucose molecule.

Pyruvate Kinase Covalent Modification Enzyme has its activity regulated also by covalent modification. In response to glucagon, a hormone that raises blood glucose levels, the enzyme is phosphorylated by protein kinase A and rendered less active. Covalent modification is probably a more primitive mode of control than allosterism.

Pyruvate Kinase Deficiency Second most common cause of enzyme deficiency-related hemolytic anemia (Glucose 6-phosphate dehydrogenase deficiency is the most common)

Lactate Dehydrogenase Pyruvate + NADH + H+ = Lactate + NAD+ When oxygen is in short supply (e.g., skeletal muscle under rigorous exercise), lactate is the end product of glycolysis, rather than pyruvate. This regenerates NAD+ for the G3P dehydrogenase reaction.

Overall Net Reaction (Anaerobic Glycolysis) A total of 4 ATP is generated, but two ATP are used to phosphorylate glucose and F6P, so the net ATP yield is 2. Glucose + 2 ADP + 2Pi = 2 lactate + 2 ATP

Intramolecular Redox There is no net oxidation as glucose is converted to two molecules of lactic acid. There is, however, intramolecular redox, resulting from a redistribution of oxygen. In glucose each C atom is bonded to an O. In lactic acid, one C has two O, one has one O, and one has none.