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Glycolysis Chapter 17 Dr. M. khalifeh Coupling of Production and Use of Energy • ATP is an essential high energy bond-containing compound • It does not store energy, it transfer it • (electric current does not store energy) • A lot of negative charges make it unsuitable for storage (repulsion) • Fat and carbohydrate store energy • Hydrolysis of ATP to ADP produce energy • Phosphorylation of ADP to ATP require energy • Supplied from oxidation of nutrients Dr. M. khalifeh ATP • Why is ATP less stable, charge-wise, than ADP? • Four –ve charges on ATP and 3 on ADP, therefore • ATP is less stable due to electrostatic repulsion. • Energy must be expended to put an additional negatively charged phosphate on ADP Dr. M. khalifeh Organophosphate as an energy source • Organophosphates release higher energy when hydrolyzed than ATP (Table 15.1) • We can use the energy release from hydrolysis of these compound to phosphorylate ADP to ATP Dr. M. khalifeh The Overall Pathway of Glycolysis • Glycolysis is the first stage of glucose metabolism • One molecule of glucose is converted to pyruvate which gives rise to 2 molecules of ATP via substrate level phosphorylation. • Note that all of the steps of glycolysis are enzymatically catalyzed. • It plays a key role in the way organisms extract energy from nutrients • Once Pyruvate is formed, it has one of several fates Dr. M. khalifeh Fates of Pyruvate From Glycolysis Dr. M. khalifeh The Reactions of Glycolysis • Phosphorylation of glucose to give glucose-6phosphate • Isomerization of glucose-6-phosphate to give fructose-6-phosphate • Phosphorylation of fructose-6-phosphate to yield fructose-1,6-bisphosphate • Cleavage of fructose-1,6,-bisphosphate to give glyceraldehyde-3-phosphate and dihydrooxyacetone phosphate • Isomerization of dihyroxyacetone phosphate to give glyceraldehyde-3-phosphate Dr. M. khalifeh The Reactions of Glycolysis (Cont’d) • Oxidation of glyceraldehyde-3-phosphate to give I,3-bisphosphoglycerate • Transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP to give ATP • Isomerization of 3-phosphoglycerate to give 2phosphoglycerate • Dehydration of 2-phosphoglycerate to give phosphoenolpyruvate • Transfer of a phosphate group from phosphoenolpyruvate to ADP to give pyruvate Dr. M. khalifeh Glycolysis phases • The pathway of glycolysis can be seen as consisting of 2 separate phases. • the first is the chemical priming phase requiring energy in the form of ATP: • 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). • the second is considered the energy-yielding phase: • F1,6BP is degraded to pyruvate, with the production of • 4 equivalents of ATP and 2 equivalents of NADH. Dr. M. khalifeh Conversion of Glucose to Glyceraldehyde-3-Phosphate • In step 1 of glycolysis, glucose is phosphorylated to give glucose-6-phosphate • The reaction is endergonic, as it is driven by the free energy of ATP hydrolysis Dr. M. khalifeh The Hexokinase Reaction • The phosphorylation by hexokinase converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. • Glucokinase is the form of the enzyme found in hepatocytes Dr. M. khalifeh Conversion of Glucose to Glyceraldehyde-3Phosphate • The second step is the Isomerization of glucose-6phosphate to fructose-6-phosphate • The C-1 aldehyde of glucose-6-phosphate is reduced to a hydroxyl group • The C-2 hydroxyl group is oxidized to give the ketone group of fructose-6-phosphate Dr. M. khalifeh Conversion of Glucose to Glyceraldehyde-3-Phosphate • Fructose-6-phosphate is then phosphorylated again to generate fructose-1,6-bisphosphate • This is the second reaction to be coupled with ATP hydrolysis Dr. M. khalifeh Phosphofructokinase is a key regulatory enzyme • Phosphofructokinase (PFK): • Exists as a tetramer and subject to allosteric feedback regulation • The tetramer is composed of L and M subunits • M4, M3L, M2L2, ML3, and L4 all exist. Combinations of these subunits are called Isozyme • Muscles are rich in M4; the liver is rich in L4 • ATP is an allosteric effector; • high levels inhibit the enzyme • low levels activate it • Fructose-1,6-bisphosphate is also an allosteric effector Dr. M. khalifeh Conversion of Glucose to Glyceraldehyde-3-Phosphate • Fructose-1,6-bisphosphate is split into • two three carbon fragments • Reaction catalyzed by Aldose • Side chains of an essential Lys and Cys play key roles in catalysis Dr. M. khalifeh Conversion of Glucose to Glyceraldehyde-3-Phosphate • In step 5, dihydroxyacetone phosphate (DHAP) is converted to glyceraldehyde-3-phosphate • These compounds are trioses • This reaction has small +G (0.58kcal/mol-1) • Remember that glycolysis has several reactions that have very negative G values, and drive other reactions to completion Dr. M. khalifeh Summary • In the first stages of glycolysis, glucose is converted to two molecules of glyceraldehyde-3-phosphate • The key intermediate in this series of reactions is fructose-1,6-bisphosphate. • The enzyme that catalyzes this reaction, phosphofructokinase, is subject to allosteric control Dr. M. khalifeh Phase II starts (payoff phase) Glyceraldehyde-3-Phosphate is Converted to Pyruvate • The first reaction that begins the conversion to pyruvate involves the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate • This reaction involves addition of a phosphate group, as well as an electron transfer reaction from glyceraldehyde-3phosphate to NAD+ • The oxidizing agent, NAD+, is reduced to NADH • Mixed anhydride of two acid by loss of water Dr. M. khalifeh Oxidation and Phosphorylation Reaction Dr. M. khalifeh Cysteine’s Role in Glyceraldehyde-3-phosphate Dehydrogenase Dr. M. khalifeh Glyceraldehyde-3-Phosphate to Pyruvate • 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate • This step involves reaction in which ATP is produced by phosphorylation of ADP • 1,3-bisphosphoglycerate transfers a phosphate group to ADP. This is known as substrate level phosphorylation. • Reaction is catalyzed by phosphoglycerate Kinase • This reaction is the sum of the endergonic phosphorylation of ADP and the exergonic hydrolysis of the mixed phosphate anhydride • The standard free energy of the hydrolysis rxn is more negative than the standard free energy of the hydrolysis of the new phosphate group being formed • Hydrolysis of 1,3-bisphosphoglycerate yield more negative free energy than ATP hydrolysis Dr. M. khalifeh Glyceraldehyde-3-Phosphate to Pyruvate Dr. M. khalifeh Glyceraldehyde-3-Phosphate to Pyruvate • The next step involves the transfer of 3-phosphoglycerate to 2-phosphoglycerate • This reaction is catalyzed by phosphoglyceromutase Dr. M. khalifeh Glyceraldehyde-3-Phosphate to Pyruvate • Next, 2-phosphoglycerate loses one molecule of water, producing Phosphoenolpyruvate • It is just dehydration rxn • Enolase catalyzes the reaction and requires a Mg as cofactor • Phosphoenolpyruvate contains a high energy bond Dr. M. khalifeh Glyceraldehyde-3-Phosphate to Pyruvate • Phosphenolpyruvate (PEP) transfers its phosphate group to ADP, producing ATP and pyruvate • G of hydrolysis of PEP is more than that of ATP (-61.9kJ mol-1 vs. -30.5kJ mol-1) • Reaction is catalyzed by pyruvate kinase • Allosteric enzyme Dr. M. khalifeh Control Points in Glycolysis Allosteric enzymes Hexokinase Allosteric enzymes Phosphofrectokinase Lack of NAD+ mean storage is fully loaded Allosteric enzymes Pyruvate Kinase Dr. M. khalifeh Summary • In the final stages of glycolysis, two molecules of pyruvate are produced for each molecule of glucose that entered the pathway • These reactions involve electron transfer, and the net production of two ATP for each glucose • There are three control points in the glycolytic pathway Dr. M. khalifeh Anaerobic Metabolism of Pyruvate • Under anaerobic conditions, the most important pathway for the regeneration of NAD+ is reduction of pyruvate to lactic acid • Lactate dehydrogenase (LDH) is a tetrameric isoenzyme consisting of H and M subunits; H4 predominates in heart muscle, and M4 in skeletal muscle Dr. M. khalifeh NAD+ Needs to be Recycled to Prevent Decrease in Oxidation Reactions Dr. M. khalifeh Alcoholic Fermentation • Two reactions lead to the production of ethanol: • Decarboxylation of pyruvate to acetaldehyde • Reduction of acetaldehyde to ethanol • Pyruvate decarboxylase catalyzes the first reaction • This enzyme require Mg2+ and the cofactor Thiamine pyrophosphate (TPP) • Alcohol dehydrogenase catalyzes the conversion of acetaldehyde to ethanol Dr. M. khalifeh Structures of Thiamine and TPP Dr. M. khalifeh Summary • Pyruvate is converted to lactate in anaerobic tissues, such as actively metabolizing muscle. NAD+ is recycled in the process • In some organisms, pyruvate is converted to ethanol in a process requiring thiamine pyrophosphate as a coenzyme Dr. M. khalifeh The Energy Derived from Glucose Oxidation • Aerobic glycolysis of glucose to pyruvate, requires two ATP to activate the process, with production of four ATP and two NADH. Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ Dr. M. khalifeh Glycolysis NADH • The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation, producing either two or three equivalents of ATP • Depending upon whether the glycerol phosphate shuttle or the malate-aspartate shuttle is used to transport the electrons from cytoplasmic NADH into the mitochondria. Dr. M. khalifeh Complete energy yield from Glycolysis • The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is, therefore, either 6 or 8 moles of ATP. • Released or stored Dr. M. khalifeh