Lecture 3: Glycolysis, Krebs Cycle, PDH Complex, and ETS Pathways PDF

Glycolysis,PDHcomplex,krebs cycle,and ETS pathways By :Elias T/mariam (BSc,MSc) 1 Outline of the lecture  Glycolysis  Pyruvate dehydrogenase complex  Krebs cycle  Electron transport chain; oxidative phosphorylation; ATP yields 2  Glycolysis  Essentiall...

Glycolysis,PDHcomplex,krebs cycle,and ETS pathways By :Elias T/mariam (BSc,MSc) 1 Outline of the lecture  Glycolysis  Pyruvate dehydrogenase complex  Krebs cycle  Electron transport chain; oxidative phosphorylation; ATP yields 2  Glycolysis  Essentially all living cells use GLYCOLYSIS as their main pathway for glucose metabolism.  It occurs in the cytosol and is also called the Embden-Meyerhof pathway.  Glycolysis is the conversion of glucose to pyruvate. It is also used for the conversion of glucose to lactate when pyruvate is converted to lactate (e.g. during anaerobic glycolysis).  There are 10 enzyme steps involved. Three of these steps are irreversible.  In the presence of oxygen, pyruvate enters mitochondria and is further metabolized to acetyl- Coenzyme A (acetyl-CoA), which is then metabolized further by the Krebs cycle.  In the absence of oxygen, anaerobic glycolysis occurs and the pyruvate is converted to lactate (catalyzed by lactate dehydrogenase, LDH) in the cytosol.  Glycolysis produces ATP, which is useful for cells to survive low oxygen conditions, where mitochondrial oxidative phosphorylation is slow. However, much less ATP is produced by glycolysis from one glucose molecule than is produced if the pyruvate was further metabolized by the Krebs cycle.  Many cancer cells produce lactate even in the presence of oxygen. This is called aerobic glycolysis. It is clear from more recent research also that many normal cells (for example red blood cells, proliferating T-lymphocytes, brain cells, also produce lactate even in the presence of oxygen, and 3 therefore aerobic glycolysis is not confined to cancer cells.  Glycolysis  The overall pathway of glycolysis is: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O  The first step is the conversion of glucose (which enters cells from the bloodstream/ extracellular environment) to glucose-6-phosphate, catalyzed by hexokinase.This step uses one ATP molecule: Glucose + ATP Glucose-6-phosphate + ADP The phosphorylation prevents the glucose-6-phosphate from leaving the cells and the reaction is irreversible. Hexokinase is an important step in glycolysis. It is a regulatory step for glycolysis; glucose-6- phosphate can be used for various purposes (glycolysis, pentose phosphate pathway, glycogen synthesis, gluconeogenesis). In some cells, e.g., liver, pancreas, there is an important type (isoform) of hexokinase called glucokinase which has a very important function as a blood glucose “sensor.” 4 5  GLYCOLYS 6  Glycolysis  The next step (isomerization) converts glucose-6-phosphate to fructose-6-phosphate. Enzyme: phosphoglucose isomerase.  The fructose-6-phosphate is then converted to fructose-1,6-bisphosphate by phosphofructokinase (PFK): fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADP This step is also important because it is a major regulatory step in glycolysis. It uses up another ATP, so that by now already the energy from 2 ATP molecules have been used for each glucose molecules metabolized. This step is also irreversible. *The initial steps of glycolysis, therefore, use 2 ATP molecules per glucose molecule and are “investments” to get the pathway to the next stages, 7 where eventually more ATP will be synthesized than used up.  Glycolysis  Fructose-1,6-bisphosphate is then cleaved into two 3-carbon molecules: glyceraldehyde-3-phosphate dehydrogenase and dihydroxyacetone phosphate. The enzyme is aldolase (also called fructose-1,6- bisphosphate aldolase).  The two 3-carbon molecules, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate dehydrogenase, are both triose phosphates, and are isomers of each other. They are interconverted to each other by triose phosphate isomerase. The conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate is favoured, and the glyceraldehyde-3-phosphate is further metabolised by glycolysis.  From this step onwards, there are two 3-carbon molecules for each glucose molecule.  The next step is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and uses NAD+ to oxidize glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate and NADH:  NAD+ + glyceraldehyde-3-phosphate + phosphate 1,3-bisphosphoglycerate + NADH + H+ This is important because it generates NADH, which can be used to make ATP. 8  Glycolysis  The next step, catalyzed by phosphoglycerate kinase, is the first step in which ATP is made. This is an example of substrate-level phosphorylation, in which ATP is made from ADP directly in an enzymatic reaction. This is distinct from the way ATP is produced by oxidative phosphorylation, from reduced molecules (NADH and FADH), via the electron transport chain. 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP  This means that 2 ATP molecules are made for each glucose molecule at this step, because two 1,3-bisphosphoglycerate molecules are derived from one glucose molecule.  Since 2 ATP molecules were used up in the hexokinase and phosphofructokinase steps, the net yield of ATP so far is zero (2 ATP used, 2 ATP made). 9  Glycolysis  The 3-phosphoglycerate is then isomerized to 2-phosphoglycerate by the enzyme, phosphoglycerate mutase.  Following this, 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP). The enzyme is enolase. Phosphoenolpyruvate contains a high energy enol-phosphate linkage, which is even more high in energy than the terminal phosphodiester bond in ATP. In the next step, this energy from the phosphoenol group of PEP is transferred to ADP to make ATP.  PEP is then converted to pyruvate, the final product of glycolysis, in the presence of oxygen. In anaerobic conditions, lactate is the final product of glycolysis. The phosphate group of the phosphoenolpyruvate is transferred in this reaction, catalyzed by pyruvate kinase, to ADP to form another ATP molecule: phosphoenolpyruvate + ADP pyruvate + ATP This is another example of substrate-level phosphorylation, where ATP is made at the reaction level and not via reducing equivalents through oxidative phosphorylation. This step makes another 2 ATP molecules per original glucose molecule. This now means that a net of 2 ATP molecules are made from one glucose molecule by glycolysis. 10  Glycolysis  Under low oxygen (anaerobic) conditions, the pyruvate cannot enter mitochondria and enter the Krebs cycle and undergo oxidative phosphorylation, because this process needs oxygen.  Therefore, the pyruvate is converted to lactate in the cytosol by the enzyme, lactate dehydrogenase (LDH). This involves reducing pyruvate to lactate using NADH. This step uses the NADH generated in an earlier step (glyceraldehyde-3-phosphate dehydrogenase step) to regenerate NAD+ and maintain a stable redox state of the cytoplasm. 11  Glycolysis  In the presence of enough oxygen, the pyruvate formed in the cytosol during glycolysis can enter mitochondria and be used, via the Krebs cycle and oxidative phosphorylation, to make ATP.  The net yield of ATP from glycolysis during aerobic conditions is higher that 2 ATP molecules per glucose because the reducing equivalents from the NADH generated during glycolysis (at the glyceraldehyde-3-phosphate step) can be used to make ATP via oxidative phosphorylation. This will be discussed in the next lecture.  When lactate is the product of glycolysis, the NADH produced at the glyceraldehyde dehydrogenase step is used up in the conversion of pyruvate to lactate (by lactate dehydrogenase). This regenerates NAD+ for re-use. In this case, therefore, NADH does not enter mitochondria for ATP production. So, glycolysis that produces lactate produces a net of only 2 ATP molecules.  Some tissues, for example skeletal muscle, may use anaerobic glycolysis on occasions when the demand for ATP exceeds the capacity of mitochondrial ATP production to supply ATP (due to limited supply of oxygen, for example during intense exercise).  Red blood cells lack mitochondria and so produce lactate from glycolysis. Other tissues, for example brain, also produce lactate normally. We will return to these issues later and discuss the idea that the classical idea of cytosolic pyruvate being the end product of glycolysis and the source of mitochondrial acetyl-CoA might need to be revised. 12 Glyceraldehyde-3-phosphate dehydrogenase step 13  Glycolysis: pyruvate may be converted to lactate (in anaerobic conditions) or enter mitochondria to be converted to acetyl-CoA, which then enters the Krebs cycle (citric acid cycle). 14  Adenylate Kinase  Cells generally maintain high ATP : AMP ratios in order to keep the ATP supply available for metabolic and biological functions. The ATP concentration may be 100 times higher than the AMP concentration.  The ubiquitous enzyme, adenylate kinase, catalyses the following reaction: 2ADP ATP + AMP * Adenylate kinase relays information about the energy status of a cell to other molecular systems, in particular AMP-activated protein kinase (AMPK). 15  Summary of Glycolysis  There are 10 enzyme-catalyzed steps in glycolysis from glucose to pyruvate, and 11 steps from glucose to lactate.  Glycolysis to pyruvate produces a net of 2 ATP molecules per glucose molecule plus 2 NADH molecules per glucose molecule.  The first step (hexokinase) is irreversible and also glucose-6-phosphate cannot leave the cell, because phosphorylated molecules do not diffuse out of cells well. This is true for all intermediates except pyruvate- they are all phosphorylated!  The first phase of glycolysis actually uses up ATP (2 ATP per glucose molecule), rather than makes ATP. This phase “sets up” the pathway for subsequent net ATP production in the later steps of glycolysis.  Know the steps where ATP is used and where ATP is made.  Know the step (glyceraldehyde-3-phosphate dehydrogenase) where NADH is formed.  Glycolysis to lactate produces a net of 2 ATP molecules per glucose molecule and ZERO NADH, because the NADH is used to convert pyruvate to lactate (lactate dehydrogenase step).  One of the main functions of glycolysis is to produce energy in the form of ATP phosphodiester bonds from energy stored in the glucose molecule. Another function is to produce pyruvate for further ATP production in mitochondria.  The phosphofructokinase step (fructose-6-phosphate to fructose-1,6-bisphosphate) is the step in glycolysis that commits the pathway to glycolysis, since the first irreversible step (hexokinase) produces glucose-6-phosphate, which can be diverted to other pathways (e.g. pentose phosphate pathway).  Summary of Glycolysis  The ATPs produced in glycolysis are mad by substrate-level phosphorylation, rather than by oxidative phosphorylation, which occurs in mitochondria via production of reducing equivalents (FADH 2 and NADH) and the electron transport pathway.  Understand: Aerobic respiration (oxidative glycolysis), anaerobic glycolysis, aerobic glycolysis.  There are 3 irreversible steps in glycolysis, where there is a significant negative Gibbs free energy. These are: Hexokinase (HK) Phosphofructokinase (PFK) Pyruvate kinase (PK)  These steps are also rate-limiting (rate-controlling) steps, where flux through the pathway is regulated.  Because glycolysis is irreversible at 3 steps, gluconeogenesis (the production of glucose from precursor molecules such as pyruvate, glycerol, alanine, lactate and others) cannot simply be a reversal of glycolysis: different enzymes catalyse the conversions in the in the opposite direction at these irreversible steps. (e.g. hexokinase step is irreversible, so a different enzyme (glucose-6-phosphatase) converts glucose-6- phosphate to glucose. Pyruvate Dehydrogenase Complex  Inside the mitochondrion, pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase complex. The net reaction is: pyruvate + NAD+ + CoA → Acetyl-CoA + NADH + H+ + CO2  This step is irreversible.  It involves the oxidative decarboxylation of pyruvate  The PDH complex consists of three catalytic enzymes: E1: Pyruvate dehydrogenase E2: An acetyltransferase (dihydrolipoamide acetyltransferase) E3: Another dehydrogenase (dihydrolipoamide dehydrogenase) These enzymes are arranged exquisitely in the multienzyme complex such that the substrates for one enzyme is passed directly to the next enzyme in the sequence, in a series of well controlled and efficient steps. Pyruvate Dehydrogenase Complex  The E1 component of the PDH complex is thiamine (Viatmin B1)-dependent.  The E2 component has bound lipoamide, a derivative of the vitamin, lipoic acid.  Coenzyme A, a sulfur-containing molecule, is derived from another vitamin, pantothenic acid (pantothenate).  The E3 is a flavoprotein (contains FAD). FAD is derived from Vitamin B2 (riboflavin).  The PDH complex is even more sophisticated because it contains other proteins and enzymes, including:  A structural protein, E3-binding protein (E3BP)  Two regulatory protein kinases (pyruvate dehydrogenase kinase) which phosphorylate the pyruvate dehydrogenase (E1) at serine residues. Phosphorylation decreases activity of the E1 dehydrogenase.  A regulatory protein phosphatase, pyruvate dehydrogenase phosphatase, which removes the phosphates added by the kinases. Dephosphorylation increases activity of the E1 dehydrogenase.  It is even more complex than this, because there are 4 different isoforms of the protein kinases and 2 isoforms of the protein phosphatase. Pyruvate Dehydrogenase Complex  In the first step (catalyzed by E1), pyruvate is first decarboxylated to release carbon dioxide, then there is reduction and acetylation of the lipoamide component of E2.  In the second step, the acetyl moiety is transferred from E2 to Coenzyme A ( a substrate of the reaction), to form acetyl-CoA.  At this stage, the acetyl-CoA has been produced as well as carbon dioxide, but the lipoamide prosthetic group is in a reduced form.  To regenerate the lipoamide in E2 to its original state, it transfers electrons to the E3 component. E3 contains FAD. Thus reduces the FAD to FADH2.  To return E3 to its original state, the FAD must be regenerated. This is achieved by E3, which transfers hydrogens from FAD to NAD+ to produce NADH.  The NADH can then enter the electron transfer chain to be used for ATP synthesis by oxidative phosphorylation.  decarboxylase FADH2 + NAD+ -> FAD + NADH + H+ dehydrogenase Acetyl transferase Pyruvate Dehydrogenase Multienzyme Complex Pyruvate Dehydrogenase Complex  The acetyl-CoA produced from the pyruvate can now enter the Krebs cycle.  Further energy has been “tapped” from the pyruvate molecule as reducing equivalents in the form of NADH, and these reducing equivalents can, in turn, be “tapped” as electrochemical energy, which can be “tapped” eventually as phosphodiester bond energy of ATP.  The structure/composition of the pyruvate dehydrogenase complex varies from tissue to tissue due to different modifications (e.g. phosphorylation) of its components and different isoforms of its proteins. This allows integration of metabolism between different tissues and exquisite control of central metabolism according to the needs of specific tissue and needs of the body as a whole.  What happens in thiamine (Vitamin B1) deficiency to the intermediates of glycolysis? ATP yield for oxidative metabolism of glucose ATP yield for oxidative metabolism of glucose Krebs Cycle Steps 1 and 2: Citrate synthase converts oxaloacetate and acetyl-CoA (which contains the atoms and bonds from the original glucose molecule) to citrate and aconitase converts citrate to isocitrate. CoA citrate synthase H20 oxaloacetate aconitase citrate isocitrate Steps 3 and 4: Isocitrate dehydrogenase converts isocitrate to alpha-ketoglutarate, converting NAD + to NADH. Alpha-ketoglutarate dehydrogenase complex then converts alpha-ketoglutarate to succinyl-CoA, using Coenzyme A and NAD+ as substrates, generating carbon dioxide and NADH. isocitrate NAD+ Isocitrate NADH dehydrogenase CO2 CoA alpha-ketoglutarate alpha-ketoglutarate dehydrogenase CO2 NAD+ NADH succinyl-CoA Steps 5 and 6: Succinyl-CoA synthetase (also called succinyl thiokinase) converts succinyl-CoA to succinate: this is a substrate-level phosphorylation that generates GTP, which is converted to ATP. Succinate dehydrogenase, a component of Complex II of the electron transport chain, containing FAD, fumarate Succinyl-CoA synthetase Succinate CoA dehydrogenase FADH2 FAD succinate Pi GTP GDP Succinyl-CoA ADP ATP Steps 7 and 8: Fumarase converts fumarate to malate. Then malate dehydrogenase converts malate to oxaloacetate, generating NADH, and providing oxaloacetate for another cycle of the Krebs cycle. NADH Oxaloacetate combines malate with acetyl-CoA to form dehydrogenase citrate, and begin another NAD round of the Krebs cycle. oxaloacetate malate fumarase H2 0 fumarate The Krebs Cycle  The Krebs cycle (citric acid cycle; tricarboxylic acid cycle) consists of 8 enzyme-catalyzed steps.  Most of the energy from the glucose molecule still has to be “tapped” by the time glucose has been converted to acetyl-CoA, the product of the pyruvate dehydrogenase complex in mitochondria.  Most of the energy from glucose (or other food fuels, including fats) is “tapped” by the Krebs cycle as NADH or FADH2 reducing equivalents: these can then be used to generate ATP from the electron transport chain/ oxidative phosphorylation.  There is one substrate-level phosphorylation, at the succinyl-CoA synthetase (succinyl thikinase) step. This step generates guanosine triphosphate (GTP) from GDP. The GTP formed can be converted to ATP by the enzyme, nucleotide diphosphate kinase: GTP + ADP  ATP + GDP The Krebs Cycle: Where is the energy?  Steps in the Krebs cycle that “tap” energy from the glucose molecule:  NADH is produced at 3 steps:  Isocitrate dehydrogenase  Alpha-ketoglutarate dehydrogenase  Malate dehydrogenase  FADH2 is produced at one step:  Succinate dehydrogenase  Substrate-level phosphorylation forms ATP (via GTP) at one step:  Succinyl-CoA synthetase  Electron transport chain and oxidative phosphorylation  When glucose has been metabolized through glycolysis/ pyruvate dehydrogenase complex/ Krebs cycle, the only “real” ATP has come from substrate-level phosphorylation (2 steps in glycolysis and 1 step in Krebs cycle).  Most of the energy tapped from the glucose molecule is in the form of reducing equivalents, as NADH and FADH2. This “redox” energy has yet to be converted to the chemical energy of ATP.  The reducing equivalents from glycolysis, pyruvate dehydrogenase complex and Krebs cycle are directed to the electron transport chain, which converts the reducing energy to a proton gradient across the inner mitochondrial membrane. This creates a proton motive force across the membrane. Peter Mitchell proposed this idea in 1961 and it has been proven to be correct. It is known and the Chemiosmotic Theory.  The protons are pumped by the components of the electron transport chain from the mitochondrial matrix to the space between the inner and outer mitochondrial membranes.  Electron transport chain and oxidative phosphorylation  The electron transport chain components are embedded in the inner mitochondrial membrane, though ubiquinone (Coenzyme Q) and cytochrome c is highly mobile and cytochrome c is a soluble protein.  There are four large multi-subunit complexes, Complexes I, II, III and IV. The ATP synthase (also known as the F0F1-ATPase, which converts ADP and inorganic phosphate to ATP, is also known as Complex V. Complex I: (NADH-Q oxidoreductase) Complex II: (Q-cytochrome c oxidoreductase) Complex III: (succinate- Q reductase) Complex IV: (cytochrome c oxidase)  Proton pumping across the membrane occurs with Complex I, III and IV.  Flavoproteins (FMN/FAD); iron-Sulphur proteins; heme proteins; coenzyme Q; are involved in transfer of electrons down a gradient, terminating in reduction of oxygen to form water.  Electron transport chain and oxidative phosphorylation  More protons are pumped form NADH than for FADH2 oxidation, so more ATPs are made from NADH.  Uncouplers such as dinitrophenol and UCP1 (uncoupling protein 1) “uncouple” electron transport from ATP formation by dissipating the proton gradient.  The electron transport chain (especially Complex I and III) is the main source of reactive oxygen species (ROS) in a cell and these, if excessive, can cause damage to cellular components. Electron transport chain and oxidative phosphorylation Electron transport chain and oxidative phosphorylation Electron transport chain and oxidative phosphorylation