Lecture 3 Carbohydrate Metabolism PDF

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 3110 BIOCHEMISTRY II – Intermediary Metabolism LECTURE 3: CARBOHYDRATE METABOLISM – GLYCOLYSIS AND GLUCONEOGENESIS  METABOLISM - REVIEW METABOLISM is a series of interconnect...

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 3110 BIOCHEMISTRY II – Intermediary Metabolism LECTURE 3: CARBOHYDRATE METABOLISM – GLYCOLYSIS AND GLUCONEOGENESIS  METABOLISM - REVIEW METABOLISM is a series of interconnected chemical reactions occurring within a cell and the chemical compounds involved in it are termed as METABOLITES. The enzymatic reactions are organized into discreet pathways which proceed in a stepwise manner, transforming substrates into end products through many specific chemical intermediates. Metabolic pathways can be of the following types: LINEAR (E.g. Glycolysis) CYCLIC (E.g. Citric acid cycle) SPIRAL (E.g. Biosynthesis of Fatty Acids) Metabolic pathways serve 2 functions: Generation of energy to drive vital functions. Synthesis of biological molecules.  METABOLIC PATHWAYS CATABOLIC ANABOLIC PATHWAYS PATHWAYS Are involved in oxidative breakdown of Are involved in the synthesis of larger complexes. compounds. They are usually exergonic in nature They are usually endergonic in nature.  CHARACTERISTICS OF METABOLISM 1. Metabolic pathways are irreversible. 2. Every metabolic pathway has a committed first step. 3. All metabolic pathways are regulated. 4. Metabolic pathways in eukaryotic cells occur in specific cellular locations. Gluconeogenesis  GLYCOLYSIS – Glucose Catabolism Glycolysis comes from a merger of two Greek words: ❑Glykys = sweet ❑Lysis = breakdown/ splitting It is also known as the Embden-Meyerhof-Parnas pathway or EMP pathway. INTRODUCTION GLYCOLYSIS is the sequence of 10 enzyme-catalyzed reactions that converts glucose into pyruvate with simultaneous production of ATP. In this oxidative process, 1 mol of glucose is partially oxidised to 2 moles of pyruvate. This major pathway of glucose metabolism occurs in the cytosol of all cells. This unique pathway occurs aerobically as well as anaerobically & doesn’t involve molecular oxygen.  It also includes the formation of Lactate from Pyruvate. The glycolytic sequence of reactions differs from species to species only in the mechanism of its regulation & in the subsequent metabolic fate of the pyruvate formed. In aerobic organisms, glycolysis is the prelude to the Citric acid cycle and electron transport chain (ETC). Glycolysis is the central pathway for glucose catabolism.  Extracellular matrix & cell wall polysaccharides. Synthesis of structural polymers Oxidation via pentose phosphate pathway Glycogen, Ribose-5- Glucose Starch, phosphate Sucrose storage Oxidation via glycolysis Major pathways of glucose utilization. Pyruvate  Phosphoglucoisomerase Phosphoglycerate kinase. Mg 2+ Phosphoglycerate mutase Overview of the Glycolytic pathway TWO PHASES OF GLYCOLYSIS Glycolysis leads to the breakdown of 6-C glucose into two molecules of 3-C pyruvate. The enzyme-catalyzed reactions are bifurcated (split) or categorised into 2 phases: 1. Phase 1- preparatory phase [requires ATP] 2. Phase 2- payoff phase [produces ATP!] PREPARATORY PHASE It consists of the first 5 steps of glycolysis in which the glucose is enzymatically phosphorylated by ATP to yield Fructose-1,6- biphosphate. This fructose-1,6-biphosphate is then split in half to yield 2 molecules of 3-carbon containing Glyceraldehyde-3- phosphate/dihydroxyacetone phosphate. Thus the first phase results in cleavage of the hexose chain. This cleavage requires an investment of 2 ATP molecules to activate the glucose molecule and prepare it for its cleavage into a 3-carbon compound.  THE PREPARATORY PHASE OF GLYCOLYSIS Phosphoglucoisomerase  PAYOFF PHASE This phase constitutes the last 5 reactions of Glycolysis. This phase marks the release of ATP molecules during the conversion of Glyceraldehyde-3-phosphate to 2 moles of Pyruvate. Here 4 moles of ADP are phosphorylated to ATP. Although 4 moles of ATP are formed, the net result is only 2 moles of ATP per mole of Glucose oxidised, since 2 moles of ATP are utilised in Phase 1.  THE PAYOFF PHASE OF GLYCOLYSIS Phosphoglycerate kinase. Mg 2+ Phosphoglycerate mutase STEPWISE EXPLANATION OF GLYCOLYSIS  STEP 1: PHOSPHORYLATION Glucose is phosphorylated by ATP to form glucose 6-phosphate. This is an irreversible reaction & is catalysed by hexokinase. Thus the reaction can be represented as follows: 6  STEP 2: ISOMERISATION It is a reversible rearrangement of the chemical structure of carbonyl oxygen from C1 to C2, forming a Ketose from the Aldose. Thus, isomerisation of the aldose Glucose 6-phosphate gives the ketose, Fructose-6-phosphate. 1 Aldose Ketose 2  STEP 3: PHOSPHORYLATION Here the Fructose-6-phosphate is phosphorylated by ATP to fructose-1,6-bisphosphate. (Makes molecule symmetrical) This is an irreversible reaction and is catalysed by phosphofructokinase enzyme. 1st committed step in the pathway!  STEP 4: BREAKDOWN This six-carbon sugar is cleaved to produce two 3-C molecules: glyceraldehyde-3-phosphate (GAP) & dihydroxyacetone phosphate(DHAP). This reaction is catalyzed by Aldolase. 6C 3C 3C  STEP 5: ISOMERIZATION Dihydroxyacetone phosphate is oxidised to form Glyceraldehyde-3- phosphate. We now have 2 molecules of Glyceraldehyde-3-phosphate. This reaction is catalyzed by the triose phosphate isomerase enzyme.  STEP 6 2 molecules of Glyceraldehyde-3-phosphate are oxidised, extracting high- energy electrons, which are picked up by the electron carrier NAD+, producing NADH. The enzyme Glyceraldehyde-3-phosphate dehydrogenase catalyzes the the addition of a second phosphate to Glyceraldehyde 3-phosphate creating 1,3- bisphosphoglycerate.  STEP 7 The transfer of high-energy phosphate group that was generated earlier to ADP, form ATP. This phosphorylation i.e. addition of phosphate to ADP to give ATP is termed as substrate level phosphorylation as the phosphate donor is the substrate 1,3-bisphosphoglycerate (1,3-BPG). The product of this reaction is 2 molecules of 3- phosphoglycerate.  STEP 8 The remaining phosphate-ester linkage in 3-phosphoglycerate, is moved from carbon 3 to carbon 2 ,because of relatively low free energy of hydrolysis, to form 2-phosphoglycerate(2-PG). 1 1 2 2 3 3  STEP 9: DEHYDRATION OF 2-PG This is the second reaction in glycolysis where a high-energy phosphate compound is formed. Enolase causes 2-phosphoglycerate to lose water from its structure. This is a dehydration reaction, resulting in the formation of a double bond that increases the potential energy in the remaining phosphate bond and produces phosphoenolpyruvate (PEP). This is a reversible reaction.  STEP 10: TRANSFER OF PHOSPHATE FROM PEP to ADP This last step is the irreversible transfer of high energy phosphoryl group from phosphoenol pyruvate to ADP. This reaction is catalyzed by pyruvate kinase. This is the 2nd substrate-level phosphorylation reaction in glycolysis which yields ATP. This is a non-oxidative phosphorylation reaction. Thus the simultaneous reactions involved in glycolysis are: Glucose is oxidized to Pyruvate NAD⁺ is reduced to NADH ADP is phosphorylated to ATP Fates of Pyruvate Entry into the citric acid cycle. Conversion to fatty acid or ketone bodies Conversion to lactate. Conversion to ethanol. OVERALL BALANCE SHEET OF GLYCOLYSIS Each molecule of glucose gives 2 molecules of Glyceraldehyde-3- phosphate. Therefore, the total input of all 10 reactions can be summarized as a net equation: Glucose+ 2Pi+ 2ADP+ 2NAD⁺ 2Pyruvate+ 2NADH+ 2ATP+ 2H⁺ + 2H₂O  ENERGY YIELD IN GLYCOLYSIS: STEP NO. REACTION CONSUMPTION of ATP GAIN of ATP 1 Glucose glucose-6- 1 - phosphate 3 Fructose-6-phosphate 1 - fructose-1,6- biphosphate 7 1,3-diphosphoglycerate - 1x2=2 3- phosphoglycerate 10 Phosphoenolpyruvate - 1x2=2 pyruvate 2 4 Net gain of ATP=4 -2= 2  Regulation of Glycolysis Three allosteric enzymes regulate and catalyse the irreversible reactions which regulate glycolysis: 1. Hexokinase (and glucokinase): hexokinase is inhibited by glucose 6-phosphate. This enzyme prevents the accumulation of glucose 6-phosphate due to product inhibition. Glucokinase specifically phosphorylates glucose. 2. Phosphofructokinase (PFK) is the most important regulatory enzyme in glycolysis. Catalyzes the rate limiting committed step. This enzyme is an allosteric enzyme – regulated by allosteric effectors. 3. Pyruvate kinase: also regulates glycolysis. This enzyme is inhibited by ATP.  GLUCONEOGENESIS The formation of glucose or glycogen from non- carbohydrate precursors is called gluconeogenesis. Importance of Gluconeogenesis: Maintain blood glucose concentration during fasting, starvation & limited carbohydrate intake. This is necessary, especially for the nervous system & erythrocytes.  Importance of gluconeogenesis Failure of gluconeogenesis is usually fatal. Hypoglycemia causes brain dysfunction, which can lead to coma & death Maintain adequate concentration of intermediates of the citric acid cycle Clear the product of metabolism of other tissue from blood. Example-Lactate produced in muscle & RBC, glycerol is continuously produced in adipose tissue. Excessive gluconeogenesis occurs in critically ill patients in response to injury & infection, contributing to hyperglycemia.  Salient features Substrate: Product: Glucose ✓ Glucogenic amino acid Site : ✓ Lactate Liver (90%) ✓ Glycerol Kidney (10-40%) ✓ Fatty acid Intestine ✓ Pyruvate Compartment : Cytoplasm & ✓ Intermediates of TCA cycle mitochondria ✓ Propionates Nature : Anabolic Glucogenic Amino Acids  Steps of Gluconeogenesis It involves glycolysis, the citric acid cycle and some other reactions Glycolysis and gluconeogenesis share the same pathway but in opposite directions Three irreversible reactions prevent simple reversal of glycolysis. The reactions are catalysed by: Hexokinase, Phosphofructokinase and Pyruvate kinase Irreversible reaction of glycolysis must be bypassed. - 3 reactions are highly thermodynamically favourable (irreversible in vivo). - Different enzymes in the different pathways.  Steps of Gluconeogenesis The reactions are reversed as follows- 1. Reaction catalysed by pyruvate kinase: Reversal involves two endothermic reactions a) Pyruvate carboxylase in mitochondria catalyzes the carboxylation of pyruvate to oxaloacetate (requires biotin and ATP). Oxaloacetate must be converted to malate to exit the mitochondria (uses 2NADH). b) Phosphoenolpyruvate carboxykinase in cytosol catalyses decarboxylation & phosphorylation of oxaloacetate to phosphoenolpyruvate (requires GTP).  Steps of Gluconeogenesis 2. Reaction catalyzed by phosphofructokinase: The conversion of fructose 1,6- bisphosphate to fructose-6-phosphate is catalyzed by fructose 1,6- bisphosphatase. 3. Reaction catalyzed by hexokinase: The conversion of glucose-6- phosphate to glucose is catalyzed by glucose-6-phosphatase. It is present in the liver & kidney but absent from muscle, which cannot export glucose into the bloodstream.  Regulation of gluconeogenesis Glucagon: affects glucose metabolism, specifically by increasing gluconeogenesis and decreasing glycolysis Epinephrine: augments hepatic glucose production by stimulating glycogenolysis and gluconeogenesis. Enzymes: ▪ Step 10: Pyruvate carboxylase and PEP carboxykinase ▪ Step 3: Fructose 1,6 bis-phosphatase ▪ Step 1: Glucose 6 phosphatase Formation of one molecule of glucose from pyruvate requires 4 ATP, 2 GTP, and 2 NADH. It is expensive.  Cori Cycle/ Lactic Acid Cycle Glucose is formed from 2 groups of compounds via gluconeogenesis ▪ Amino acids involve in direct net conversion ▪ Product of metabolism of glucose in tissue Thus, lactate, formed by glycolysis in skeletal muscle & RBC, is transported to the liver & kidneys where it reforms glucose Which again becomes available via the circulation for oxidation in the tissues. This process is known as Cori cycle.  Glucose-alanine cycle In fasting state there is considerable output of alanine from skeletal muscle. It is formed by transamination of pyruvate produced by glycolysis of muscle glycogen & is exported to the liver. There after transamination back to This glucose-alanine cycle pyruvate & becomes a substrate provides an indirect way of for gluconeogenesis. utilizing muscle glycogen to maintain blood glucose in the fasting state  Disorders of Carbohydrate Metabolism The metabolism of the carbohydrates galactose, fructose, and glucose are linked through interactions between different enzymatic pathways, and disorders that affect these pathways may have symptoms ranging from mild to severe or even life- threatening: 1.Lactose intolerance 2.Congenital sucrase-isomaltase deficiency (CSID) 3. Galactosemia 4. Glycogen storage diseases 5. Hereditary fructose intolerance 6. Diabetes mellitus 1. Lactose Intolerance Lactose of milk cannot be hydrolysed due to a deficiency of lactase. Accumulation of lactose in the intestinal tract, which is “osmotically active” & holds water, producing diarrhoea. Accumulated lactose is also fermented by intestinal bacteria which produce gas & other products, producing flatulence & abdominal pain. 2. Congenital sucrase-isomaltase deficiency (CSID) Rare inherited metabolic disorder characterized by the deficiency or absence of the enzymes sucrase and isomaltase. Symptoms may include watery diarrhoea resulting in abnormally low levels of body fluids (abdominal swelling (and/or abdominal discomfort). In addition, some affected infants may experience malnutrition, resulting from malabsorption of essential nutrients, and/or a delay in growth and weight gain. 3. Galactosemia Galactosemia is a rare, hereditary disorder of carbohydrate metabolism that affects the body’s ability to convert galactose (a sugar contained in milk, including human mother’s milk) to glucose (a different type of sugar). The disorder is caused by a deficiency of the enzyme galactose 1 phosphate uridylyl transferase ( which is vital to this process). A metabolite that is toxic to the liver and kidneys builds up. The metabolite also damages the lens of the eye, causing cataracts. 4. Glycogen Storage Diseases Glycogen storage diseases are carbohydrate metabolism disorders that occur when there is a defect in the enzymes that are involved in the metabolism of glycogen, often resulting in growth abnormalities, weakness, a large liver, low blood sugar, and confusion. Glycogen storage diseases are caused by the lack of an enzyme needed to change glucose into glycogen and break down glycogen into glucose. Typical symptoms include weakness, sweating, confusion, kidney stones, a large liver, low blood sugar, and stunted growth. Examples of Glycogen Storage Diseases 5. Hereditary fructose intolerance Hereditary fructose intolerance is a carbohydrate metabolism disorder that is caused by a lack of the enzyme needed to metabolize fructose. Very small amounts of fructose cause low blood sugar levels and can lead to kidney and liver damage. Fructose intolerance disorders occur when parents pass the defective genes that cause these disorders on to their children. Typical symptoms include low blood sugar, sweating, confusion, and kidney damage. 6. Diabetes mellitus Diabetes mellitus refers to a group of diseases that affect how your body uses blood sugar (glucose). Glucose is vital to your health because it's an important source of energy for the cells that make up your muscles and tissues. It's also your brain's main source of fuel. Chronic diabetes conditions include type 1 diabetes and type 2 diabetes Potentially reversible diabetes conditions include prediabetes and gestational diabetes. Prediabetes occurs when your blood sugar levels are higher than normal, but not high enough to be classified as diabetes. END OF LECTURE 3