1Biochemistry I Fall Term, 2003November 10 & 12, 2003Lecture 29: GlycolysisAssigned reading in Campbell: Chapter 14Recommended Problems from Campbell Ch. 14: 5, 7, 13, 18, 22, 24, 29, 37, 42, 49, 50.Key Terms:Anaerobic glycolysisInduced fitIsozymeNAD+ & NADHRegulated stepsSubstrate-level phosphorylationLinks:(I) Review Quiz on Lecture 29 concepts:(I) Glycolysis Pathway Summary: Class handout on the 10 reactions in the pathway.(S) Free Energy Profile of Glycolysis: in the Human Red Blood Cell(S) Glycolysis Intermediates: Chime images of the 10 reactions in the pathway.(S) Hexokinase Induced Fit: Chime images of the two conformations.(S) Protein Structure Examples: Chime images of Triose Phosphate Isomerase, PyruvateKinase, and Lactate Dehydrogenase.Central Pathways of Energy Production2Intracellular locations• Glycolysis: cytosol• Fatty Acid Oxidation: matrix space of mitochondria• TCA Cycle: matrix space of mitochondria• Oxidative Phosphorylation: Inner membrane of mitochondriaFree Energy ManagementEnergy is stored in the following ways:• High energy chemical species (e.g. phosphoanhydride bonds in ATP)• Redox reactions produce NADH• Membrane potentials (concentration gradient and voltage difference)Utilized in the following ways:• Chemical synthesis reactions (e.g. protein synthesis, DNA & RNA synthesis)• Mechanical work (e.g. solute transport & muscle function)• Electrical work (e.g. ion transport & nerve conduction)Chapters 14-16 of Campbell describes many (interesting) details of the enzyme mechanisms. Forthe pathways described in these chapters, you should know the following:• Input substrates (and their structures) to the pathway.• Output products (and their structures) from the pathway.• Regulatory steps and how they are regulated.• General enzymatic nomenclature.• Cellular location of each pathway.You will not be required to know the details of each mechanistic step and there will be littleemphasis on detailed knowledge of the structures of the intermediates in the pathway. However,some knowledge of the structure of the intermediates will certainly help you to understand thechemistry associated with each enzymatic transformation. You should become familiar with eachof the enzymatic steps in these pathways at a level illustrated below for glyceraldehyde-3-phosphate dehydrogenase. In this example NAD+ could be considered either as a substrate or acoenzyme.3The lectures on these chapters will focus on several key steps in each pathway, e.g. there are fourto be emphasized in glycolysis. Substrate(s) of the reaction Glyceraldehyde-3-phosphate and Pi Name of the enzyme Glyceraldehyde-3-phosphate dehydrogenase Name of the cofactor bound to the enzyme NAD+ Product(s) of the reaction NADH and 1,3-bisphosphoglycerate Type of reaction Oxidation and substrate-level phosphorylation.Enzyme Nomenclature:Kinase: transfers a phosphate group from ATP (e.g. hexokinase, galactose kinase, pyruvatekinase)Isomerase: converts one isomer to another (e.g. phosphoglucoisomerase, triosephosphateisomerase)Aldolase: catalyzes aldol condensation (e.g. aldolase, functions in reverse in glycolysis)Dehydrogenase: removes hydrogens by oxidation. Usually require NAD+ or FAD ascofactors/cosubstrates). In some cases results in formation of a "high-energy" phosphatebond by substrate level phosphorylation (e.g. glyceraldehyde-3-phosphate dehydrogenase).Mutase: group transfer enzyme. Common use is to move phosphates to different positions onsugars (e.g. phosphoglycerate mutase, glucose-1-phosphate mutase).Enolase: converts C=C group to alcohol. No change in oxidation state. Functions in reversedirection in glycolysis (enolase) and forward direction in fatty acid oxidation and in the citricacid cycle (fumarase).Synthase: (also known as synthetase). Usually an enzyme that combines two things to makea new compound. (e.g. citrate synthase, succinyl CoA synthetase). In the citric acid cyclecitrate synthase runs in the forward direction while succinyl CoA runs in the reversedirection.ATPase: Hydrolyzes ATP to ADP and Pi. This reaction runs in reverse in FoF1 ATPase togenerate ATP using the free energy of the proton gradient.4General Features of Glycolysis:• Glycolysis is found in all living organisms.• Input: Glucose, 2 ATP, 4 ADP, 2 NAD+.• Functions under aerobic or anaerobic conditions.• Output (aerobic): 2 NADH, pyruvate, 2 ADP, 4 ATP.• Output (anaerobic): lactate, 2 ADP, 4 ATP.• Net energy gain under aerobic conditions: 2 ATP and 2 NADH.• Rapid production of ATP from glucose.• Lactose, sucrose, and glycogen enter the pathway above the regulatory step (PFK).This is an example of the convergence of diverse substrates onto a common pathway.• All of the intermediates are phosphorylated.Four Key Enzyme Reactions 1. Hexokinase (HK): Step 1• Glucose transported into the cell by facilitated diffusion cannot diffuse out as G-6-P.• Phosphorylates other hexoses.• Example of induced fit by glucose binding.• Transfer of ATP phosphate to water is negligible.Water is excluded from the active site by the conformational change.• Net energetics are favorable ∆ G°' = -16.7 kJ/mol. 2. Phosphofructokinase (PFK): Step 3• Inhibited by ATP, citrate, and phosphoenolpyruvate (PEP).• Activated by ADP, AMP, cAMP, fructose-2,6-bisphosphate (F2,6P).• Tetrameric allosteric enzyme with two ATP binding sites (one allosteric; the other at theactive site).1. T state (inactive) is stabilized by ATP, citrate, phosphoenolpyruvate.2. R state (active) is stabilized by ADP, AMP, cAMP, F2,6P.5• ATP is buffered in cells by the enzyme, adenylate kinase, that converts 2ADP to ATP +AMP. Therefore levels of ATP do not change much. Consequently, the higher levels ofAMP and ADP during ATP demand largely regulate PFK activity.• The enzyme catalyzing the back reaction (fructose-1,6-bisphosphatase) is inhibited byAMP and F2,6P. The combined regulation of the forward and reverse pathways can resultin a 100 fold change in the flux through this step.Note that after Step 3 glycolysis has used two ATPs for each glucose that enters theglycolytic pathway. 3. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH): Step 6• Oxidation of the aldehyde, producing 3-phosphoglycerate, is exergonic.• Substrate-level phosphorylation is endergonic.1. The "high energy" bond formed is a mixed acid anhydride.2. Pi (HPO42-) is the substrate.HAsO42- (arsenate)