Lecture 2- Glycolysis - Metabolic Fates of Pyruvate - PDF

Glycolysis and Metabolic fates of pyruvate Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences Division of Structural & Computational Biology Singapore 637551 [email protected]  State of reduction of carbon atoms in biomolecules...

Glycolysis and Metabolic fates of pyruvate Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences Division of Structural & Computational Biology Singapore 637551 [email protected]  State of reduction of carbon atoms in biomolecules more oxidised Cate More reduced state Less reduced state Fats Carbohydrates Carbonyls Carboxyls Carbon dioxide In living systems most of the energy needed to drive biosynthetic reactions is derived from the oxidation of organic substrates. Oxygen, the ultimate electron acceptor for aerobic organisms, is a strong oxidant; it has the tendency to attract electrons, becoming reduced in the process, e.g.: Lgaine ; gets reduced becomes oxidized becomes reduced Garett & Grisham: Biochemistry 4th edition  The energy levels of electrons ⚫ Energy is the capacity to cause change ⚫ Potential energy is the energy that matter has because of its location or structure ⚫ The electrons of an atom have potential energy due to their distance from the nucleus ⚫ Changes in potential energy occur in steps of fixed amounts ⚫ An electron’s energy level is correlated with its average distance from the nucleus A ball bouncing down a flight of stairs can come to rest only on each step, not between steps. Third shell (highest energy level in this model) Second shell (higher Energy energy level) absorbed First shell (lowest Energy level) Energy lostreleased Atomic nucleus © 2017 Pearson Education, Ltd.  Stepwise Energy Harvest via NAD+ becomes oxidized becomes reduced In cellular respiration, glucose and other organic molecules are broken down in a series of steps. Electrons from organic compounds are usually first transferred to NAD+, a coenzyme 2 e− + 2 H+ 2 e− + H+ NAD+ NADH H+ Dehydrogenase Reduction of NAD+ 2[H] H+ (from food) Oxidation of NADH Nicotinamide Nicotinamide lose H (reduced form) (oxidized form) © 2017 Pearson Education, Ltd.  Overview of carbohydrate digestion The major carbohydrates in the diet are starch, lactose, and sucrose. The starches amylose and amylopectin are polysaccharides composed of hundreds to millions of glucosyl units linked together through α-1,4- and α-1,6-glycosidic bonds (Fig. right). Lactose is a disaccharide composed of glucose and galactose, linked together through a β-1,4-glycosidic bond. Sucrose is a disaccharide composed of glucose and fructose, linked through an α-1,2-glycosidic bond. The digestive processes convert all of these dietary carbohydrates to their constituent monosaccharides by hydrolyzing glycosidic bonds between the sugars. Glucose is the universal fuel for human cells and the source of carbon for the synthesis of most other compounds. Every human cell type uses glucose to obtain energy. The release of the hormones insulin and glucagon by the pancreas aids in the body’s use and storage of glucose. Other dietary sugars (mainly fructose and galactose) are converted to glucose or to intermediates of glucose metabolism. These monosaccharides are absorbed from the intestine, enter the blood, and travel to the tissues, where they are metabolized (Fig. right). C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Overview of carbohydrate metabolism of energy s~ torage ~ monosacc. polysaccharide After glucose is transported into cells, it is phosphorylated by a hexokinase to form glucose 6- phosphate. Glucose 6-phosphate can then enter several metabolic pathways. The three that are common to all cell types are glycolysis, the pentose phosphate pathway, and glycogen synthesis (Fig. right). Important. The phosphate group in glucose 6- phosphate (G6-P) is completely ionized at physiological pH, giving it an overall negative charge. Major pathways of glucose metabolism. Since the plasma membrane is impermeable to charged molecules, G6-P cannot enter into the cells from the blood stream. In tissues, fructose and galactose are converted to intermediates of glucose metabolism. Thus, the fate of these sugars parallels that of glucose (Fig. right). Overview of fructose and galactose metabolism. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Overview of glycolysis and the TCA cycle loxtI : need ATP first then ATP generates ~ gain Glycolysis (from the Greek glyk-, sweet, and lysis, splitting) begins er with the phosphorylation of glucose to glucose 6–phosphate (glucose + My I NAD 6-P) by hexokinase. In subsequent steps of the pathway, one glucose 6-P molecule is oxidized to two pyruvate molecules with generation of two molecules of nicotinamide adenine dinucleotide (NADH). cleared 2 A net generation of two molecules of ATP occurs through direct 2 lothand inpolysis transfer of high-energy phosphate from intermediates of the pathway to adenosine diphosphate (ADP) (substrate-level phosphorylation). Glycolysis occurs in the cytosol and generates cytosolic NADH. Because NADH cannot cross the inner mitochondrial membrane, its reducing equivalents are transferred to the electron-transport chain by either the malate–aspartate shuttle or the glycerol 3-phosphate shuttle. Pyruvate is then oxidized completely to CO2 by pyruvate dehydrogenase and the tricarboxylic acid (TCA) cycle inside the mitochondrion. Complete aerobic oxidation of glucose to CO2 can generate approximately 30 to 32 mol of ATP per mole of glucose. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Energy Investment Phase 2 MPT : X+E & formul Glyceraldehyde 3-phosphate (G3P) ATP Glucose Fructose ATP Fructose Glucose 6-phosphate 6-phosphate 1,6-bisphosphate ADP ADP Isomerase Hexokinase Phosphogluco- Phospho- Aldolase Dihydroxyacetone isomerase fructokinase phosphate (DHAP) Glucose metabolism begins with transfer of a phosphate from ATP to glucose to form glucose 6-P. Phosphorylation of glucose commits it to metabolism within the cell because glucose 6-P cannot be transported back across the plasma membrane. The phosphorylation reaction is irreversible under physiologic conditions because the reaction has a high- negative free, ΔG0′. Phosphorylation does not, however, commit glucose to glycolysis. This reaction of the first ATP investment involves nucleophilic attack of the C6-OH of glucose on the electrophilic terminal (γ) phosphate of ATP. Magnesium ion is required ↓ because the reactive form of ATP is its chelated complex with Mg2+. Mg2+ partially 2 neutralizes the1 negative charges of the oxygen atoms, making the γ-phosphorus atom more accessible for nucleophilic attack, and thus a better electrophile. (irreversible xH) inATP exergonic ext = process gie Never forget me! © 2017 Pearson Education, Ltd. D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019) C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Energy Investment Phase Glyceraldehyde 3-phosphate (G3P) ATP Glucose Fructose ATP Fructose Glucose 6-phosphate 6-phosphate 1,6-bisphosphate ADP ADP Isomerase Hexokinase Phosphogluco- Phospho- Aldolase Dihydroxyacetone isomerase fructokinase phosphate (DHAP) ClowElse ,itall low kn a ~ very : Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a family of tissue-specific isoenzymes that differ in their kinetic properties. The iso-enzyme found in liver and β-cells of the pancreas has a much higher Km than other hexokinases and is called Lesser glucokinase. In many cells, some of the hexokinase is bound to porins in the outer mitochondrial membrane, which gives these enzymes first access to newly synthesized ATP as it exits the mitochondria. affinite for glurge , birds giurse Glucose 6-P is isomerized to fructose 6-phosphate (fructose 6-P) and subsequently cleaved better ehigh a into two three-carbon fragments. The isomerization is essential for the subsequent cleavage of the bond between carbons 3 and 4. & & max speed & low Iglucose)reyokinage always bird e glucose while giurokinase takes only , , lover some glusse a its speed is binding of e enzyme better e to its substrate lower e Km value , i © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Isomerization of glucose 6-phosphate to fructose 6-phosphate 1 Never forget me! 2 2 C, 10 alcohol & publated I 7 activate of 23 ↑ isomerisaty Recall that the open-chain form of glucose has an aldehyde group at carbon 1, whereas the open-chain form of fructose has a keto group at carbon 2. Thus, the isomerization of glucose 6-phosphate to fructose 6- phosphate is a conversion of an aldose into a ketose. The reaction catalyzed by phosphoglucose isomerase includes additional steps because both glucose 6-phosphate and fructose 6-phosphate are present primarily in the cyclic forms. The enzyme must first open the six-membered ring of glucose 6-phosphate, catalyze the isomerization, and then promote the formation of the five-membered ring of fructose 6-phosphate. In summery, the isomerization reaction is necessary because the “moving” of the carbonyl from C-1 to C-2 creates a new primary alcohol function at C-1, which becomes easily phosphorylated, activation of C-3, facilitating the C-C bond cleavage in the 4th step of glycolysis. J. M. Berg, J. L. Tymoczko & L. Stryer, Biochemistry 7th edition  Reaction of the PFK-1 within the Energy Investment Phase Glyceraldehyde 3-phosphate (G3P) ATP Glucose Fructose ATP Fructose Glucose 6-phosphate 6-phosphate 1,6-bisphosphate ADP ADP Isomerase Hexokinase Phosphogluco- Phospho- Aldolase Dihydroxyacetone isomerase fructokinase phosphate (DHAP) The next step of glycolysis (reaction 3) is the phosphorylation of fructose 6-P to fructose 1,6-bisphosphate (fructose 1,6-bisP). The reaction involves the same nucleophilic substitution chemistry we saw for the hexokinase (reaction 1). Here in reaction 3, the C1-OH of F6P is the nucleophile that attacks the electrophilic γ-phosphate of ATP. The reaction catalyzed by phosphofructokinase-1 (PFK-1), is generally considered the first committed step of the pathway and is thermodynamically and kinetically irreversible. Therefore, PFK-1 irrevocably commits glucose to the glycolytic pathway. PFK-1 is a regulated enzyme in cells, and its regulation controls the entry of glucose into glycolysis. Like the hexokinase, it exists as tissue-specific isoenzymes whose regulatory properties match variations in the role of glycolysis in different tissues. © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Aldol cleavage within the Energy Investment Phase Glyceraldehyde 3-phosphate (G3P) ATP Glucose Fructose ATP Fructose Glucose 6-phosphate 6-phosphate 1,6-bisphosphate ADP ADP Isomerase Hexokinase Phosphogluco- Phospho- Aldolase Dihydroxyacetone isomerase fructokinase phosphate (DHAP) Fructose 1,6-bisP is cleaved into two phosphorylated three-carbon compounds (triose phosphates) by the aldolase. Dihydroxyacetone phosphate (DHAP) is isomerized to glyceraldehyde 3-P, which is a triose phosphate. The aldolase is named for the mechanism of the forward reaction, which is an aldol cleavage, and the mechanism of the reverse reaction, which is an aldol condensation. © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Mechanism for the fructose-1,6 bisphosphate aldolase reaction Never forget me! The aldolase activates the substrate for cleavage by nucleophilic attack on the keto carbon at position 2 with a lysine ε-amino group in the active site. This is facilitated by a protonation of the carbonyl oxygen by an active site (aspartate). The resulting carbinolamine undergoes dehydration to give an iminium ion, or protonated Schiff base (Schiff base: imine; R2C=NR’). A Schiff base is a nucleophilic addition product between an amin group and a carbonyl group. A reto-aldol reaction then cleaves the protonated Schiff base into an enamine plus GAP. The enamine is protonated to give another iminium ion (protonated Schiff base; reaction 4), which is then hydrolyzed off the enzyme to give the second product, DHAP. Ni The Schiff base intermediate is advantageous in this reaction because it can delocalize electrons. The positively charged iminium ion is thus a better electron acceptor than a ketone carbonyl, facilitating retro- aldol reactions. This reaction also demonstrates why it was important to isomerize G6P to F6P in reaction 2. If glucose had not been isomerized to fructose (moving the carbonyl from C-1 to C-2), then the aldolase reaction would have given two- and four-carbon fragments, instead of the metabolically equivalent three-carbon fragments. D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019)  Energy Payoff Phase 2 ATP 2 ATP 2 NADH 2 H2O Glyceraldehyde 2 ADP (x2) 3-phosphate (G3P) 2 NAD+ + 2 H+ 2 ADP 2 2 2 2 2 Triose Phospho- Phospho- Enolase Pyruvate phosphate 2 Pi glycerokinase glyceromutase kinase dehydrogenase 1,3-Bisphospho- 3-Phospho- 2-Phospho- Phosphoenol- Pyruvate glycerate glycerate glycerate pyruvate (PEP) In the energy payoff phase of the glycolytic pathway, glyceraldehyde 3-P is oxidized and phosphorylated so that subsequent intermediates of glycolysis can donate phosphate to ADP to generate ATP. The first reaction in this sequence, catalyzed by glyceraldehyde 3-P dehydrogenase (a Triose phosphate dehydrogenase), is really the key to the pathway. This enzyme oxidizes the aldehyde group of glyceraldehyde 3-P to an enzyme-bound carboxyl group and transfers the electrons to NAD+ to form NADH. The oxidation step is dependent on a cysteine residue at the active site of the enzyme, which is followed by the process of substrate-level phosphorylation (the formation of a high-energy phosphate bond where none previously existed, without the use of oxygen). In the next (7th) reaction, the phosphate in this bond is transferred to ADP to form ATP by phosphoglycerate kinase. The energy of the acyl phosphate bond is high enough (~10 kcal/mol) so that transfer to ADP is an energetically favorable process. Another product of this reaction is 3-phosphoglycerate. © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Reactions of the Glyceraldehyde 3-P dehydrogenase 2 ATP 2 ATP 2 NADH 2 H2O Glyceraldehyde 2 ADP 3-phosphate (G3P) 2 NAD+ + 2 H+ 2 ADP 2 2 2 2 2 Triose Phospho- Phospho- Enolase Pyruvate phosphate 2 Pi glycerokinase glyceromutase kinase dehydrogenase 1,3-Bisphospho- 3-Phospho- 2-Phospho- Phosphoenol- Pyruvate glycerate glycerate glycerate pyruvate (PEP) The glyceraldehyde 3-P dehydrogenase glyceraldehyde (a Triose phosphate dehydrogenase) oxidizes the aldehyde group of glyceraldehyde A 3-P to an enzyme-bound carboxyl group and transfers the electrons to NAD+ to form NADH. The oxidation step is dependent on a cysteine residue at the active site of the enzyme, which forms a high-energy thioester bond during (deprotonated the course of the reaction. The high-energy intermediate immediately accepts an inorganic phosphate to form the high-energy acyl phosphate bond in 1,3-bisphosphoglycerate (High-energy phosphates are indicated by the red squiggles in the Figure), releasing the product from the cysteine & form high-evegy residue on the enzyme. This high-energy phosphate bond is the start of this ester bond substrate-level phosphorylation (the formation of a high-energy phosphate bond where none previously existed, without the use of oxygen). inorganic a phosphat © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Reactions in steps 8-10 of the Energy Payoff Phase 2 ATP 2 H2O 2 ADP 2 2 2 2 Phospho- Enolase Pyruvate glyceromutase kinase 3-Phospho- 2-Phospho- Phosphoenol- Pyruvate glycerate glycerate pyruvate (PEP) To transfer the remaining low-energy phosphoester on 3-phosphoglycerate to ADP, it must be converted into a high-energy bond. This conversion is accomplished by moving the phosphate to the second carbon (forming 2- phosphoglycerate) and then removing water to form phosphoenolpyruvate (PEP). The enolphosphate bond is a high-energy bond (its hydrolysis releases approximately 14 kcal/mol of energy), so the transfer of phosphate to ADP by pyruvate kinase is energetically favorable. This final reaction converts PEP to pyruvate. © 2017 Pearson Education, Ltd. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Phases of the glycolytic pathway The glycolytic pathway, which cleaves Energy Investment Phase 1 mol of glucose to 2 mol of the three- carbon compound pyruvate, consists of a Glucose preparative phase (energy investment phase) and an ATP-generating phase (energy payoff phase). In the initial preparative phase of glycolysis, glucose is phosphorylated twice by ATP and 2 ATP used 2 ADP + 2 P cleaved into two triose phosphates. In the ATP-generating phase, glyceraldehyde 3-phosphate (glyceraldehyde 3-P) (a triose phosphate) is oxidized by NAD+ and phosphorylated using inorganic Energy Payoff Phase phosphate. The high-energy phosphate bond generated in this step is transferred 4 ADP + 4 P 4 ATP formed to ADP to form ATP. The remaining phosphate is also rearranged to form another high-energy phosphate bond that is transferred to ADP. Because 2 mol of triose phosphate were formed, the yield from the ATP-generating phase is 4 mol 2 NAD+ + 4 e− + 4 H+ 2 NADH + 2 H+ of ATP and 2 mol of NADH. The result is a net yield of 2 mol of ATP, 2 mol of NADH, and 2 mol of pyruvate per mole of glucose. 2 Pyruvate + 2 H2O Net Glucose 2 Pyruvate + 2 H2O 4 ATP formed − 2 ATP used 2 ATP 2 NAD+ + 4 e− + 4 H+ 2 NADH + 2 H+ © 2017 Pearson Education, Ltd.  The critical reaction steps in glycolysis ~ irrevesible processes (might be regulated in glycolysis) Note the 3 large negative standard free-energy changes in the 10 reaction steps of glycolysis. ∆G values are estimated from the approximate intracellular concentrations of glycolytic intermediates in rabbit skeletal muscle. Remember: The thermodynamically favored direction of a reaction is determined by changes in both the enthalpy (H) and the entropy (S). The free energy, G = H - TS, takes both into account. The criterion for a favorable process is that free energy change ∆G = ∆H - T∆S is negative (“exergonic”), rather than positive (“endergonic”); this is one statement of the second law of thermodynamics. D. L. Nelson, M. & M. Cox, Lehninger Principles of Biochemistry, 4th edition C. M. Smith, A. D. Marks & M. A. Liebermann Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019) Coffee break  Major sites of regulation in the glycolytic pathway & by irreversible steps G GP (pdt) cot by rexokinase can committed give -he feedback step to it ~ Hexokinase and phosphofructokinase-1 are ~ - poked in mito Th in cycle the major regulatory enzymes in skeletal muscle. The activity of pyruvate dehydrogenase in the mitochondrion determines whether pyruvate is converted to lactate or to acetyl coenzyme A (acetyl-CoA). ~ The regulation shown for pyruvate kinase occurs only for the liver (L) isoenzyme. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Regulation of Phosphofructokinase-1 Phosphofructokinase-1 (PFK-1) is an allosteric enzyme that has a total of six binding sites: two are for substrates (MgATP and fructose 6-P) and four are allosteric regulatory sites. The allosteric regulatory sites occupy a different domain on the enzyme than the catalytic site. The allosteric sites for PFK-1 include an inhibitory site for MgATP, an inhibitory site for citrate, an allosteric activation site for AMP, and an allosteric activation site for fructose 2,6- bisphosphate (fructose 2,6-bisP). & very low if ATP binds to two different sites on the enzyme: the substrate-binding site and an allosteric inhibitory site. Under physiologic conditions, the ATP concentration is usually high enough to saturate the substrate-binding site and inhibit the enzyme by binding to the ATP allosteric site. This effect of ATP is opposed by AMP, which binds to a separate allosteric activator site. For most of the PFK-1 isoenzymes, the binding of AMP increases the affinity of the enzyme for fructose 6-P. At high [ATP], PFK-1 behaves cooperatively, and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoid. High [ATP] thus inhibits PFK-1, decreasing the enzyme’s affinity for fructose-6-phosphate. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition R. H. Garrett & C. M. Grisham, Biochemistry, 4th edition  Regulation of Glucose content Overview of the major pathways of glucose metabolism. Pathways for production of blood glucose are shown by dashed lines. Acetyl-CoA, acetyl coenzyme A; DHAP, dihydroxyacetone phosphate; FA, fatty acids; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid; TG, triacylglycerols. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Hormonal regulation of glycolysis ~ fact The regulation of glycolysis by allosteric activation or inhibition, or the phosphorylation/dephosphorylation of rate-limiting enzymes, is short term — that is, they influence glucose consumption over periods of minutes or hours. Superimposed on these moment-to-moment effects are slower, and often more profound, hormonal influences on the amount of enzyme protein synthesized. These effects can result in 10-fold to 20-fold increases in enzyme activity that typically occur over hours to days. Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver. These changes reflect an increase in gene transcription, resulting in increased enzyme synthesis. High activity of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the wellfed state. Conversely, gene transcription and synthesis of glucokinase, phosphofructokinase, and pyruvate kinase are decreased when plasma glucagon is high and insulin is low, for example, as seen in fasting or diabetes. R. Harvey & D. Ferrier, BIOCHEMISTRY 5th edition  Biosynthetic functions of glycolysis O ② Glycolysis, in addition to providing ATP, generates precursors for biosynthetic pathways. Intermediates of the pathway can be converted to ①ribose 5- phosphate, the sugar incorporated into nucleotides ⑤ such as ATP. Other sugars, such as UDP-glucose, mannose, and sialic acid, are also formed from intermediates of glycolysis.⑤Serine is synthesized ⑪ from 3-phosphoglycerate, and ⑪alanine from pyruvate. The ebackbone of triacylglycerols, glycerol 3-P, is derived from DHAP in the glycolytic pathway. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Anaerobic glycolysis When cells have a limited supply of oxygen (e.g., the kidney medulla), or few or no mitochondria (e.g., the red blood cell), or greatly increased demands for ATP (e.g., skeletal muscle during high-intensity exercise), they rely on anaerobic glycolysis for generation of ATP. In anaerobic glycolysis, lactate dehydrogenase oxidizes the NADH generated from glycolysis by reducing pyruvate to lactate. Because O2 is not required to reoxidize the NADH, the pathway is referred to as anaerobic. The IH] energy yield from anaerobic glycolysis (2 mol ATP per mole of glucose) is much lower than the yield from aerobic oxidation. The lactate (lactic acid) is released into the blood. Under pathologic conditions that cause hypoxia, tissues may generate enough lactic acid to cause Slow lactic acidemia. o levels buid-up of lactate , excessive low pH C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Oxidative Fates of Pyruvate and Nicotinamide Adenine Dinucleotide The NADH produced from glycolysis must be continuously reoxidized back to NAD+ to provide an electron acceptor for the glyceraldehyde 3-P dehydrogenase reaction and prevent product inhibition. Without oxidation of this NADH, glycolysis cannot continue. There are two alternate routes for oxidation of cytosolic NADH. One route is aerobic, involving shuttles that transfer reducing equivalents across the mitochondrial membrane and ultimately to the electron-transport chain and oxygen (A). The other route is anaerobic (without the use of oxygen). In anaerobic glycolysis, NADH is reoxidized in the cytosol by lactate dehydrogenase (LDH), which reduces pyruvate to lactate (B). C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Lactate dehydrogenase reaction When the oxidative capacity of a cell is limited (e.g., in the red blood cell, which has no mitochondria), the pyruvate and NADH produced from glycolysis cannot be oxidized aerobically. The NADH is therefore oxidized to NAD+ in the cytosol by reduction of pyruvate to lactate. The reduction of pyruvate is catalyzed by lactate dehydrogenase, which forms the L-isomer of lactate at pH 7: Note: The overall equilibrium of this reaction strongly favors lactate formation, as shown by the large negative standard free-energy change. D. L. Nelson, M. & M. Cox, Lehninger Principles of Biochemistry, 4th edition  Fermented milk products Yogurt and Dahi are fermented milk products produced by bacterial fermentation of milk. The bacteria, Lactobacillus and Streptocossus, used to make yogurt are known as "yogurt cultures". Fermentation of lactose by these bacteria produces lactic acid, which acts on milk proteins to give yogurt and dahi the texture and characteristic tang.  Fate of Lactate ~ no mite epyruvate Carl F. Cori (1897-1984) Gerty T. Cori (1896-1957) Lactate released from cells that undergo anaerobic glycolysis is taken up by other tissues (primarily the liver, heart, and skeletal muscle) and oxidized back to pyruvate. In the liver, the pyruvate is used to synthesize glucose (gluconeogenesis), which is returned to the blood. The cycling of lactate and glucose between peripheral tissues and liver is called the Cori cycle. The heart, with its huge mitochondrial content and oxidative capacity, is able to use lactate released from other tissues as a fuel. During exercise such as bicycle riding, lactate released into the blood from skeletal muscles in the leg might be used by resting skeletal muscles in the arm. In the brain, glial cells and astrocytes produce lactate, which is used by neurons or released into the blood. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition Voet, Voet, Pratt: Principles of BIOCHEMISTRY 3rd edition  Tissues of the eye are also partially dependent on anaerobic glycolysis The eye contains cells that transmit or focus light, and these cells, therefore, cannot be filled with opaque structures such as mitochondria or densely packed capillary beds. The corneal epithelium generates most of its ATP aerobically from its few mitochondria but still metabolizes some glucose anaerobically. Oxygen is supplied by diffusion from the air. The lens of the eye is composed of fibers that must remain birefringent to transmit and focus light, so mitochondria are nearly absent. The small amount of ATP required can be generated from anaerobic glycolysis even though the energy yield is low. The lens is able to pick up glucose and release lactate into the vitreous body and aqueous humor. It does not need oxygen and has no use for capillaries. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Anaerobic conversion of Pyruvate to Ethanol The synthesis of ethanol↑ by highly selected strains of yeast is important in the production of beer and wine. Yeast cells convert pyruvate to ethanol and CO2 and oxidize NADH to NAD+. Two reactions are required: Pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires Mg2+ and has a tightly bound coenzyme, thiamine pyrophosphate (TPP). Alcoholdehydrogenase catalyzes the reduction of acetaldehyde to ethanol. This oxidation-reduction reaction is coupled to oxidation of NADH. The sum of the glycolytic reaction and the conversion of pyruvate to ethanol is it is no MADH as L. A. Moran, H. R. Horton, K. G. Scrimgeour & M. D. Perry, Principles in Biochemistry, 5th edition paced In used D. L. Nelson, M. & M. Cox, Lehninger Principles of Biochemistry, 4th edition  TPP and its role in pyruvate decarboxylation & Nu attack & - Stabilised is complex releases ~ protons easily TPP is the coenzyme form of vitamin B1 (thiamine). The role of the functional group of thiamine pyrophosphate (the reactive carbon in the thiazolium ring is shown in red) in formation of a covalent intermediate. (A) A base on the enzyme (B) abstracts a proton from thiamine, creating a carbanion (general acid–base catalysis). (B) The carbanion is a strong nucleophile and attacks the partially positively charged keto group on the substrate. (C) A covalent intermediate is formed, which, after decarboxylation, is stabilized by resonance forms. The uncharged intermediate is the stabilized transition- state complex. L. A. Moran, H. R. Horton, K. G. Scrimgeour & M. D. Perry, Principles in Biochemistry, 5th edition D. L. Nelson, M. & M. Cox, Lehninger Principles of Biochemistry, 4th edition C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Production of Swiss Cheese Three types of bacteria are used in the production of Emmental cheese: Streptocossus salivarius, Lactobacillus, and Propionibacterium. In a late stage of cheese production, the propionibacteria consume the lactic acid excreted by the other bacteria and release acetate, propionc acid, and carbon dioxide gas. The carbon dioxide slowly forms the bubbles that develop the "eyes". The acetate and propionic acid give Swiss its nutty and sweet flavor. In general, the larger the eyes in a Swiss cheese, the more pronounced its flavor because a longer fermentation period gives the bacteria more time to act. http://en.wikipedia.org/wiki/Swiss_cheese  Fermentation Overview © 2017 Pearson Education, Ltd  Major pathways for fermentation of sugars organisms end pat Major pathways for fermentation of sugars including organisms involved and end products formed V. Müller (2001) Bacterial Fermentation. ENCYCLOPEDIA OF LIFE SCIENCES  Industrial products from fermentations V. Müller (2001) Bacterial Fermentation. ENCYCLOPEDIA OF LIFE SCIENCES  The evolutionary significance of Glycolysis ⚫ Glycolysis is the most common metabolic pathway among organisms on Earth, indicating that it evolved early in the history of life ⚫ Early prokaryotes may have generated ATP exclusively through glycolysis due to the low oxygen content in the atmosphere ⚫ The location of glycolysis in the cytosol also indicates its ancient origins; eukaryotic cells with mitochondria evolved much later than prokaryotic cells CITRIC OXIDATIVE GLYCOLYSIS PYRUVATE ACID PHOSPHORYL- OXIDATION CYCLE ATION ATP © 2017 Pearson Education, Ltd  Comparison of fermentation and respiration Glucose Glycolysis CYTOSOL Pyruvate No O2 present: O2 present: Fermentation Aerobic cellular respiration MITOCHONDRION Ethanol, Acetyl CoA lactate, or other products CITRIC ACID CYCLE © 2017 Pearson Education, Ltd  Quiz 𝟏. 𝐖𝐡𝐢𝐜𝐡 one of the following statements concerning glycolysis is correct? A. The conversion of glucose to lactate requires the presence of oxygen. B. Hexokinase is important in hepatic glucose metabolism only in the absorptive period following consumption of a carbohydrate-containing meal. C. Fructose 2,6-bisphosphate is a potent inhibitor of phosphofructokinase. D. The regulated reactions are also the irreversible reactions. E. The conversion of glucose to lactate yields two ATP and two NADH. 2. The reaction catalyzed by phosphofructokinase-1: A. is activated by high concentrations of ATP and citrate. B. uses fructose 1-phosphate as substrate. C. is the rate-limiting reaction of the glycolytic pathway. D. is near equilibrium in most tissues. E. is inhibited by fructose 2,6-bisphosphate.  Quiz 3. A major role of glycolysis is which of the following? A. To synthesize glucose B. To generate energy C. To produce FAD(2H) D. To synthesize glycogen E. To use ATP to generate heat  Quiz 4. Which one of the following organs has the highest demand for glucose as a fuel? A. Brain B. Muscle (skeletal) C. Heart D. Liver E. Pancreas The brain requires glucose because fatty acids cannot readily cross the blood–brain barrier to enter neuronal cells. Thus, glucose production is maintained at an adequate level to allow the brain to continue to burn glucose for its energy needs. The other organs listed as possible answers can switch to the use of alternative fuel sources (lactate, fatty acids, amino acids) and are not as dependent on glucose for their energy requirements as is the brain. Thank you!