Weill Cornell Medicine-Qatar Principles of Biochemistry Lecture 16a Guide PDF

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 16a Carbohydrates oxidation: Glycolysis Lecture 16b Glucose Synthesis: Gluconeogenesis Glycolysis and Gluconeogenesis: A Balancing Act Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 14: p. 510-539 Lecture 16a CARBOHYDRATES OXIDATION: GLYCOLYSIS Key topics: – Harnessing energy from glucose via glycolysis – Fermentation under anaerobic conditions DIGESTION AND ABSORPTION OF CARBOHYDRATES From Understanding Nutrition, 14th edition FOUR MAJOR PATHWAYS OF GLUCOSE UTILIZATION Storage – Can be stored in the polymeric form (starch, glycogen) – When there’s plenty of excess energy Glycolysis – Generates energy via oxidation of glucose – Short-term energy needs Pentose Phosphate Pathway – Generates NADPH via oxidation of glucose – For detoxification and the biosynthesis of lipids and nucleotides Synthesis of extracellular matrix (ECM) and Structural Polysaccharides – For example, in structure of cell walls of bacteria, fungi, and plants or in animals in cell adhesion and growth, cellular fate, and host tissue reactions 1/ Citric Acid Cycle 2/ Electron transport Chain and Oxidative Phosphorylation IMPORTANCE OF GLYCOLYSIS Glucose is an excellent fuel – Yields good amount of energy upon oxidation – Can be efficiently stored in the polymeric form – Many organisms and tissues can meet their energy needs on glucose only Glucose is a versatile biochemical precursor – Used to build the carbon skeletons of: Amino acids Membrane lipids Nucleotides in DNA and RNA Cofactors needed for the metabolism Sequence of enzyme-catalyzed reactions by which glucose is converted into pyruvate Pyruvate can be further aerobically oxidized Pyruvate can be used as a precursor in biosynthesis Some of the oxidation free energy is captured by the synthesis of ATP and NADH GLYCOLYSIS: OVERVIEW In the evolution of life, glycolysis probably was one of the earliest energyyielding pathways It developed before photosynthesis, when the atmosphere was still anaerobic Thus, the task upon early organisms was: How to extract free energy from glucose anaerobically? The solution: – First: Activate it by phosphorylation – Second: Collect energy from the highenergy metabolites generated GLYCOLYSIS HAS TWO PHASES: A PREPARATORY PHASE AND A PAYOFF PHASE The preparatory phase, steps 1 to 5, converts the sixcarbon glucose into two three-carbon units, each of them phosphorylated. In the payoff phase, steps 6 to 10, initiates the oxidation of the three-carbon units. For each molecule of glucose that passes through the preparatory phase: (a) two molecules of glyceraldehyde 3-phosphate are formed (dihydroxyacetone phosphate is isomerized to glyceraldehyde 3-phosphate); both pass through the payoff phase Input: 2 ATP 2NAD+ (b) Pyruvate is the end product of the second phase of glycolysis § For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. § For each glucose molecule, 2 NAD+ are consumed in step 6 and need to be regenerated for glycolysis to continue output: 4 ATP & 2 NADH (c) 3 irreversible reactions: are the target of tight regulation. (all the other reactions are reversible). Those very reactions are bypassed during gluconeogenesis CHEMICAL LOGIC OF GLYCOLYSIS In this simplified version of the pathway, each molecule is shown in a linear form, with carbon and hydrogen atoms not depicted, in order to highlight chemical transformations. Glucose and fructose are present mostly in their cyclized forms in solution, although they are transiently present in linear form at the active sites of some of the enzymes in this pathway. - The preparatory phase, steps 1 to 5, converts the six-carbon glucose into two three-carbon units, each of them phosphorylated. - Oxidation of the three-carbon units is initiated in the payoff phase. To produce pyruvate, the chemical steps must occur in the order shown. THE PREPARATORY PHASE OF GLYCOLYSIS (STEP 1 TO 5) REQUIRES ATP STEP 1: PHOSPHORYLATION OF GLUCOSE First priming phosphorylation of glycolysis (uses ATP) STEP 1: PHOSPHORYLATION OF GLUCOSE Rationale – Traps glucose inside the cell – Lowers intracellular glucose concentration to allow further uptake This process uses the energy of ATP Catalyzed by Hexokinase. Regulated: inhibited by the product (glucose-6-P) in all tissues, except in the liver Nucleophilic oxygen at C6 of glucose attacks the last (g) phosphate of ATP ATP-bound Mg++ facilitates this process by shielding the negative charges on ATP Highly thermodynamically favorable/irreversible – Regulated mainly by product (G-6-P) inhibition STEP 2: ISOMERIZATION OF GLUCOSE 6-PHOSPHATE TO FRUCTOSE 6-PHOSPHATE STEP 2: ISOMERIZATION OF GLUCOSE 6-PHOSPHATE TO FRUCTOSE 6-PHOSPHATE Rationale – C1 of fructose is easier to phosphorylate by PFK (next step) – Allows for symmetrical cleavage by aldolase in later step An aldose (glucose) can isomerize into a ketose (fructose) via an enediol intermediate The isomerization is catalyzed by the active-site glutamate, via general acid/base catalysis Slightly thermodynamically unfavorable/reversible – Product concentration kept low to drive forward STEP 3: PHOSPHORYLATION OF FRUCTOSE 6-PHOSPHATE TO FRUCTOSE 1-6-BISPHOSPHATE Second priming phosphorylation of glycolysis (uses ATP) STEP 3:PHOSPHORYLATION OF FRUCTOSE 6-PHOSPHATE TO FRUCTOSE 1-6-BISPHOSPHATE Rationale – Further activation of glucose – Allows for 1 phosphate/3-carbon sugar after step 4 First Committed Step of Glycolysis – fructose 1,6-bisphosphate is committed to become pyruvate and yield energy – intermediates (glucose 6 P) before this step can have multiple fates. This process uses the energy of ATP Highly thermodynamically favorable/irreversible Phosphofructokinase-1 is highly regulated – By ATP, fructose-2,6-bisphosphate, and other metabolites – Meaning of regulation: Do not burn glucose if there is plenty of ATP STEP 4: CLEAVAGE OF FRUCTOSE 1-6-BISPHOSPHATE STEP 4: CLEAVAGE OF FRUCTOSE 1-6-BIPHOSPHATE Rationale – Cleavage of a six-carbon sugar into two three-carbon sugars – High-energy phosphate sugars are three-carbon sugars The reverse process is the familiar aldol condensation Animal and plant aldolases employ covalent catalysis Fungal and bacterial aldolases employ metal ion catalysis Thermodynamically unfavorable/reversible. However, – Glyceraldehyde 3-phosphate (GAP) concentration kept low to pull reaction forward STEP 5: INTERCONVERSION OF THE TRIOSES PHOSPHATE STEP 5: INTERCONVERSION OF THE TRIOSES PHOSPHATE Rationale: – Allows glycolysis to proceed by one pathway – Aldolase (previous step) creates two triose phosphates: Dihydroxyacetone Phosphate (DHAP) and Glyceraldehyde-3-Phosphate (GAP) – Only GAP is the substrate for the next enzyme – DHAP must be converted to GAP Completes preparatory phase Thermodynamically unfavorable/reversible – GAP concentration kept low to pull reaction forward THE PAYOFF PHASE (STEP 6 TO 10) OF GLYCOLYSIS YIELDS ATP AND NADH STEP 6: OXYDATION OF GLYCERALDEHYDE 3-PHOSPHATE TO 1-3 BISPHOSPHOGLYCERATE This step uses NAD+ and produces NADH. [NAD+] in the cell is about 10 µM (much smaller than the [glucose] metabolized). Thus, the NADH formed needs to be reoxidized in NAD+ (regeneration of NAD+) for glycolysis to continue. STEP 6: OXIDATION OF GLYCERALDEHYDE 3PHOSPHATE TO 1-3 BISPHOSPHOGLYCERATE Rationale: – Generation of a high-energy phosphate compound – Incorporates inorganic phosphate – Which allows for net production of ATP (in step 7) First (of the two, the second being reaction 9) energyconserving reactions in glycolysis Oxidation of aldehyde with NAD+ gives NADH (2 NAD+ used/glucose à 2 NADH formed) Active site cysteine in G3PDH – Forms high-energy thioester intermediate – Subject to inactivation by oxidative stress Thermodynamically unfavorable/reversible. However, – Coupled to next reaction (step 7) to pull forward STEP 7: PHOSPHORYL TRANSFER FROM 1-3 BISPHOSPHOGLYCERATE TO ADP: 1ST PRODUCTION OF ATP Step 6 and 7 of glycolysis are coupled reactions in which 1,3 BPG is the common intermediate. BPG is formed in reaction 6 which is endergonic in isolation (see previous slide). The overall reaction (6+7) is exergonic STEP 7: PHOSPHORYL TRANSFER FROM 1-3 BISPHOSPHOGLYCERATE TO ADP 1ST PRODUCTION OF ATP Rationale: – Substrate-level phosphorylation to make ATP – Net production of 2 ATP/glucose 1,3-bisphosphoglycerate is a high-energy compound – can donate the phosphate group to ADP to make ATP Kinases are enzymes that transfer phosphate groups from ATP to various substrates Highly thermodynamically favorable/reversible – Is reversible because of coupling to GAPDH reaction STEP 8: CONVERSION OF 3-PHOSPHOGLYCERATE TO 2-PHOSPHOGLYCERATE: MIGRATION OF THE PHOSPHATE See similarities in reaction mechanism with phospho-gluco mutase in glycogen metabolism STEP 8: CONVERSION OF 3-PHOSPHOGLYCERATE TO 2-PHOSPHOGLYCERATE: MIGRATION OF THE PHOSPHATE Rationale: – Prepares for step 9 à Be able to form high-energy phosphate compound Mutases catalyze the (apparent) migration of functional groups One of the active site histidines is post-translationally modified to phosphohistidine Phosphohistidine donates its phosphate to oxygen of C2 before retrieving another phosphate from oxygen of C3 – 2,3-bisphosphoglycerate intermediate – Note that the phosphate from the substrate ends up bound to the enzyme at the end of the reaction Thermodynamically unfavorable/reversible – Reactant concentration kept high by phosphoglycerate Kinase to push forward STEP 9: DEHYDRATION OF 2-PHOSPHOGLYCERATE TO PHOSPHOENOLPYRUVATE STEP 9:DEHYDRATION OF 2-PHOSPHOGLYCERATE TO PHOSPHOENOLPYRUVATE Rationale – Generate a high-energy phosphate compound (this is the second energy conserving reaction, the other being step 6) 2-Phosphoglycerate is not a good enough phosphate donor – Two negative charges in 2-PG are fairly close – But loss of phosphate from 2-PG would give a secondary alcohol with no further stabilization Slightly thermodynamically unfavorable/reversible – Product concentration kept low to pull forward STEP 10: TRANSFER OF THE PHOSPHORYL GROUP FROM PHOSPHOENOLPYRUVATE TO ADP: 2ND PRODUCTION OF ATP 2nd production of ATP STEP 10: TRANSFER OF THE PHOSPHORYL GROUP FROM PHOSPHOENOLPYRUVATE TO ADP: 2ND PRODUCTION OF ATP Rationale – Substrate-level phosphorylation to make ATP – Net production of 2 ATP/glucose Loss of phosphate from PEP yields an enol that tautomerizes into ketone Tautomerization – effectively lowers the concentration of the reaction product – drives the reaction toward ATP formation Pyruvate kinase requires divalent metals (Mg++ or Mn++) for activity Highly thermodynamically favorable/irreversible – Regulated by ATP, divalent metals, and other metabolites PYRUVATE TAUTOMERIZATION DRIVES ATP PRODUCTION FREE ENERGIES OF GLYCOLYTIC REACTIONS SUMMARY OF GLYCOLYSIS Glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H20 Used: – 1 glucose; 2 ATP; 2 NAD+ Made: – 2 pyruvate Various different fates – 4 ATP Used for energy-requiring processes within the cell – 2 NADH Must be reoxidized to NAD+ in order for glycolysis to continue Glycolysis is highly regulated – Ensure proper use of nutrients – Ensure production of ATP only when needed GLYCOLYSIS IS NOT ONLY ABOUT PYRUVATE AND ENERGY FORMATION BUT IS ALSO ABOUT GLYCOLYTIC INTERMEDIATES FORMATION THAT FUNNEL INTO SEVERAL BIOSYNTHETIC PATHWAYS ENTRY OF DIETARY GLYCOGEN, STARCH, DISACCHARIDES, AND HEXOSES INTO THE PREPARATORY STAGE OF GLYCOLYSIS. Feeder pathways for glycolysis Glycogen is cleaved by glycogen phosphorylase yielding – glucose-1-phosphate – glucose-1-phosphate is then isomerized into glucose-6phosphate, that enters glycolysis Disaccharides are hydrolyzed by different enzymes in monosaccharides that enter glycolysis – Lactose gives glucose and galactose via lactase – Sucrose gives glucose and fructose via sucrase Monosaccharides fructose, galactose, and mannose – Enter glycolysis at different points (see the two important pathways of metabolism of fructose) ENTRY OF DIETARY GLYCOGEN, STARCH, DISACCHARIDES AND HEXOSES INTO THE PREPARATORY STAGE OF GLYCOLYSIS CATABOLIC FATES OF PYRUVATE Two possible catabolic fates of the pyruvate formed in glycolysis NADH must be recycled to regenerate NAD+ Cytosol Cori Cycle 1/ Under aerobic conditions, pyruvate is oxidized to acetylCoA 2/ Under anaerobic conditions or low oxygen condition (hypoxia), pyruvate is reduced to lactate or ethanol Pyruvate also serves as a precursor in many anabolic reactions (not shown here). Mitochondria (high oxidative capacity tissues i.e. cardiac muscle) FATE OF PYRUVATE: ANAEROBIC GLYCOLYSIS FERMENTATION Reduction of pyruvate to another product – (lactate in animals, ethanol in yeast) Generation of energy (2 ATP) without consuming oxygen or NAD+ No net change in oxidation state of the sugars Regenerates NAD+ for further glycolysis under anaerobic conditions The process is used (in presence of yeast or other microorganisms) in the production of food from beer to yogurt to soy sauce FATE OF PYRUVATE IN ANIMALS LACTIC ACID FERMENTATION Reduction of pyruvate to lactate (reversible) – In muscle, during strenuous exercise (O2 is not carried to the muscle fast enough to oxidize pyruvate) à lactate builds up in the muscle Generally in less than 1 minute The acidification of muscle prevents its continuous strenuous work – In RBCs (no mitochondria) à continuous lactate formation The lactate can be transported to the liver and converted to glucose there by gluconeogenesis (see Cori Cycle) – Requires a recovery time in muscle – High amount of oxygen consumption (to make ATP) to fuel gluconeogenesis (“energivore”) – To replenish muscle glycogen stores THE CORI CYCLE Metabolic cooperation between skeletal muscle and the liver: the Cori cycle. Extremely active muscles use glycogen as energy source, generating lactate via glycolysis. During recovery, some of this lactate is transported to the liver and converted to glucose via gluconeogenesis. This glucose is released to the blood and returned to the muscles to replenish their glycogen stores. The overall pathway (glucose → lactate → glucose) constitutes the Cori cycle. PATHWAYS FOR REGENERATION OF NAD+ NADH Produced in Step 6 1/ In most cells under most conditions, NADH is used through the ETC for oxidative phosphorylation in mitochondria (thus regenerates NAD+). 2/ However, some NADH is used to convert pyruvate to lactate, regenerating NAD+. Reoxidation of NADH in anaerobic glycolysis REGENERATION OF NAD+ BY LACTATE DEHYDROGENASE IN THE CYTOSOL (ANAEROBIC CONDITIONS) OR SHUTTLE SYSTEMS (FOR ETC) IN MITOCHONDRIA (AEROBIC CONDITIONS) Cytosol Normal conditions (mitochondria) Shuttle systems - Cells with no mitochondria (RBCs…) - Vigorously active muscle FATE OF PYRUVATE IN YEAST AND MICROORGANISMS: ETHANOL FERMENTATION Glucose + 2ADP + 2Pi à2 ethanol + 2 CO2 + 2ATP + 2H2O Pyruvate decarboxylase (absent in humans) uses the cofactor TPP. The CO2 produced serves in carbonation of beer or rising dough in baking… FATE OF PYRUVATE IN YEAST AND MICROORGANISMS: ETHANOL FERMENTATION Two-step reduction of pyruvate to ethanol CO2 produced in the first step is responsible for: – carbonation in beer – dough rising when baking bread Both steps require cofactors – Pyruvate decarboxylase: Mg++ and thiamine pyrophosphate – Alcohol dehydrogenase: Zn++ and NAD+ Humans do not have pyruvate decarboxylase – However, we do express alcohol dehydrogenase (ADH) for ethanol metabolism: (cytosol) - Ethanol à acetaldehyde (ADH) - Then acetaldehyde à acetate with Aldehyde dehydrogenase (ALDH). - Then acetate à acetyl-CoA à (various fates) (ER) THIAMINE PYROPHOSPHATE (TPP) IS A COMMON ACETALDEHYDE CARRIER § The thiazolium ring of TPP stabilizes carbanion intermediates by providing an electrophilic (electron deficient) structure into which the carbanion electrons can be delocalized by resonance. § Structures with this property, often called “electron sinks,” play a role in many biochemical reactions, facilitating carbon–carbon bond cleavage (adjacent to carbonyl groups, see decarboxylation reactions) and transfer of acetaldehyde from one carbon to another. Note: TPP (derived from Thiamine=Vitamin B1) deficiency (rare) leads to beriberi (accumulation of body fluids, swelling, pain, paralysis and death) and Wernicke encephalopathy. ROLE OF TPP IN DECARBOXYLATION AND OTHER REACTIONS Thiamine pyrophosphate (TPP) and its role in pyruvate decarboxylation. (a) TPP is the coenzyme form of vitamin B1 (thiamine). The reactive carbon atom in the thiazolium ring of TPP is shown in red. In the reaction catalyzed by pyruvate decarboxylase, two of the three carbons of pyruvate are carried transiently on TPP in the form of a hydroxyethyl, or “active acetaldehyde,” group (b), which is subsequently released as acetaldehyde. GLYCOLYSIS IS UNDER TIGHT REGULATION The flux of glucose through the glycolytic pathway is regulated to maintain - Constant ATP levels - Adequate supplies of glycolytic intermediates for biosynthetic pathways. This is achieved by an interplay between: - ATP consumption - NADH regeneration - Allosteric regulation of key enzymes (hexokinase, phosphofructokinase and pyruvate kinase) On a longer time scale, glycolysis is regulated by the hormones - Glucagon - Insulin - Epinephrine - Changes of gene expression of several glycolytic enzymes. An abnormal regulation of glycolysis occurs in cancer (see the Warburg effect). HIGH RATE OF GLYCOLYSIS IN TUMORS The Warburg effect Warburg observation: cancer cells show higher rates of glycolysis, relative to normal cells, with lactate fermentation, even in the presence of oxygen. Normal cells respire fully in O2 thus lowering the rate of glycolysis and oxidation of pyruvate in mitochondria (through negative regulation). The "Warburg hypothesis”: Insufficient cellular respiration caused by insult to mitochondria is at the basis of tumorigenesis (Ex: mutations in p53 à defects in mitochondrial respiration). Thus, high rate of glycolysis in cancer cells is necessary: - for high rate of ATP production* - for high rate of formation of glycolysis intermediates needed for cell proliferation In tumors, glucose transporters and most of the glycolytic enzymes are overproduced. Compounds that inhibit hexokinase, glucose 6phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it. *The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. HIGH RATE OF GLYCOLYSIS IN THE DETECTION OF CANCEROUS TISSUE BY POSITRON EMISSION TOMOGRAPHY (PET) PET scan: Indicates regions of high glucose utilization Ingestion of 18F-deoxyGlucose (FdG), uptake by Glut transporters, used as substrate by hexokinase (step 1 of glycolysis), but not by the isomerase (step 2 of glycolysis). Thus, 6-phospho FdG accumulates. Then the decay of 18F yields positrons, that are detected by the scanner Brain Computed tomography (CT scan): Location of soft tissues and bones Colored PET scan: Intensity (related to the level of FdG utilization) increases from green to red Bladder PET scan (black and white) Cancer in bones, upper spine, liver, and part of the muscles, not brain (high glucose utilization normally) and bladder (high excretion of 6-phospho FdG in urine) JC07 GLUCOSE UPTAKE IS DEFICIENT IN TYPE1 DIABETES Effect of type 1 diabetes on carbohydrate and fat metabolism in adipocytes. Normally, insulin triggers the insertion of GLUT4 transporters into the plasma membrane by the fusion of GLUT4-containing vesicles with the membrane, allowing glucose uptake from the blood. When blood levels of insulin drop, GLUT4 is resequestered in vesicles by endocytosis. In type 1 (insulin-dependent) diabetes mellitus, these normal processes are inhibited as indicated by X. The lack of insulin prevents glucose uptake via GLUT4; as a consequence, cells are deprived of glucose and blood glucose is elevated. Lacking glucose for energy supply, adipocytes break down triacylglycerols stored in fat droplets and supply the resulting fatty acids to other tissues for mitochondrial ATP production. Two byproducts of fatty acid oxidation in the liver (acetoacetate and β-hydroxybutyrate) accumulate and are released into the blood, providing fuel for the brain but also decreasing blood pH, causing ketoacidosis. The same sequence of events takes place in muscle, except that myocytes do not store triacylglycerols and instead take up fatty acids that are released into the blood by adipocytes. REGULATION OF GLYCOLYSIS BY METABOLITES Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 14: p. 533-539