Chapter 8 Glycolysis PDF

UNIT II: Intermediary Metabolism Chapter 8 Glycolysis -Pathway is occur in all tissues -Glucose broken to give energy as ATP and intermediates for other metabolic pathways -It is at the hub of CHO metabolism because all sugars, from diet or from catabolic reactions in bod...

UNIT II: Intermediary Metabolism Chapter 8 Glycolysis -Pathway is occur in all tissues -Glucose broken to give energy as ATP and intermediates for other metabolic pathways -It is at the hub of CHO metabolism because all sugars, from diet or from catabolic reactions in body, can be converted to glucose -Pyruvate is the end product of glycolysis in cells with mitochondria & an adequate supply of O2 in aerobic glycolysis. in cells with mitochonderia -Alternatively, in anaerobic glycolysis glucose is converted to pyruvate, which is reduced by NADH → lactate in cells with no mitochobnderis as RBS or in limitted supply of O2. Figure 8.9. A. Glycolysis shown as one of the essential pathways of energy metabolism. B. Reactions of aerobic glycolysis. C. Reactions of anaerobic glycolysis. IV. Transport of glucose into cells - Glucose cannot diffuse into cells, but a transport - A. Na+-independent facilitated diffusion transport - A family of 14 glucose transporters in CMs. (GLUT-1 to GLUT-14) These monomeric proteins and contain 12 transmembrane  helices -Extracellular glucose binds to transporter, which then change its conformation to transporting glucose across the CM -GLUT-1high in erythrocytes & blood brain barrier, but is low in adult muscle. -GLUT-2 in liver, kidney & β cells of pancreas. -GLUT-3 in neurons. -GLUT-4 (increased by insulin )in muscle & adipose tissue. B. Na+-monosaccharide cotransporter system - A. Na+-dependent energy requiring transport (SGLT) - Transports glucose “against” a conc. gradient i.e., from low gluc conc’s outside cell to higher conc’s within cell - movement of glucose is coupled to the conc. gradient of Na+, which is transported into cell at the same time. - occurs in intestine, renal tubules, and choroid plexus (part of blood brain barrier, which contain GLUT-1) - https://www.youtube.com/watch?v=LW_3Ji0mlEc V. Reactions of glycolysis -Conversion of gluc to pyruvate occurs in 2 stages. -1st five reactions correspond to an energy investment phase in which phosphorylated forms of intermediates are synthesized at the expense of ATP -subsequent reactions of glycolysis constitute an energy generation phase in which a net of 2 molecules of ATP are formed by substrate level phosphorylation per gluc molecule metabolized Figure 8.11 Two phases of aerobic glycolysis. A.Phosphorylation of glucose - Phosphorylated sugar polar cant diffuse out of cell→ G-6-P trapped in ell cytocol →thus committing it to further metabolism - Hexokinase phosphorylate glucose, there is four (I-IV) isozymes of it. - Hexokinases I-III *have a low Km (high affinity) for gluc.→ efficient phosphorylation & subsequent metabolism of gluc(even if of gluc are low) *have a low Vmax for gluc →cannot trap cellular phosphate as phosphorylated hexoses, or phosphorylate more sugars than cell can use *Are inhibited by reaction product, G-6-P, which accumulates when further metabolism of hexose-P is reduced 2. Hexokinase IV (Glucokinase): -In liver parenchymal cells & islet cells of pancreas -In β-cells, acts as gluc sensor, determines threshold for insulin secretion -In neurons of hypothalamus→ has arole in response to hypoglycemia. -In liver, enz facilitates gluc phosphorylation during hyperglycemia -high Km, requiring a higher gluc conc for half- saturation. → it functions only when intracellular conc of gluc in hepatocyte is high ( after consumption of a CHO-rich meal) when high levels of gluc are delivered to liver via portal vein -high Vmax, allows liver to effectively remove flood of gluc delivered by portal blood → prevents large amounts of gluc from entering systemic circulation→ minimizes hyperglycemia during absorptive period b. Regulation by fructose 6-phosphate and glucose: - Glucokinase activity is not allosterically inhibited by G-6-P as other hexokinases - It is indirectly inhibited by F-6-P (which is in equil. with G-6-P - A GKRP exists in nucleus of hepatocytes. - High F-6-P→ GK is translocated into nucleus & binds tightly to RP→ enz inactive - High gluc levels in the blood causes the release of GK from RP & enz enters cytosol where it phosphorylates gluc → G-6-P - F-1-P inhibit formation of GK-GKRP complex. B. Isomerization of G-6-P to F-6-P is catalyzed by phosphoglucose isomerase. Reaction is readily reversible & is not a rate-limiting or regulated step C. Phosphorylation of fructose 6-phosphate - Irreversible phosphorylation reaction catalyzed by phosphofructokinase-1 (PFK-1) is the most important control point & rate- limiting step of glycolysis - PFK-1 is controlled by available conc’s of substrates ATP & F-6-P, & by the following regulatory substance 1. Regulation by energy levels in cell: -Inhibited allosterically by: a)high levels of ATP, which act as an “energy- rich” signal indicating abundance of high- energy cpds. b)High levels of citrate,(intermediate in TCA cycle) → using glucose for glycogen synthesis - Activated allosterically by high conc’s of AMP, which signal that the cells’ energy stores are depleted 2. Regulation by fructose 2,6-bisphosphate - F-2,6-bisP is the most potent activator of PFK-1, it activate PFK-1 when ATP levels are high. - F-2,6-bisP is formed from F-6-P by phosphofrucokinase-2 (differe than PFK-1) - FPK-2 is bifunctional protein: Kinase: phosphorylatr F-6-P →F-2,6-bisP Phosphatase: dephosphorylate F-2,6-bisP → F-6-P - In liver, kinase domain is active if dephosphorylated and inactive if phosphorylated. - F-2,6-bisP also acts as an inhibitor of fructose 1,6-bisphosphatase - The reciprocal actions of F-2,6-bisP on glycolysis & gluconeogenesis ensure that both pathways are not fully active at the same time (Note: this would →“futile cycle” in which glucose is converted to pyruvate followed by re-synthesis of glucose from pyruvate - F-2,6-bisP is converted back to F-6-P by fructose bisphosphatase-2 a. During the well-fed state:Decreased levels of glucagon & elevated levels of insulin, as following a CHO-rich meal → cause an increase in F-2,6-bisP→ increase rate of glycolysis in the liver. -F-2,6-bisP acts as an intracellular signal, indicating high glucose -b. During starvation:- Elevated levels of glucagon & low levels of insulin, as during fasting, → decrease conc of hepatic F-2,6-bisP.→ decrease rate of glycolysis and an increase in gluconeogenesis D. Cleavage of fructose 1,6-bisphosphate - Aldolase A cleaves F-1,6-bisP to dihydroxyacetone- P (DHAP) & glyceraldehyde-3-P (GA-3P). reaction i s reversible & not regulated Note: Aldolase B in liver and kidney also cleaves F-1,6-bisP, & functions in metabolism of dietary fructose E. Isomerization of dihydroxyacetone phosphate - Triose phosphate isomerase interconverts DHAP & GA-3P. DHAP must be isomerized to GA-3P for further metabolism by glycolysis - This isomerization results in net production of 2 GA-3P from cleavage products of F-1,6-bisP F. Oxidation of glyceraldehyde 3-phosphate - by GA-3P dehydrogenase is 1st redox reaction of glycolysis (Note: because of the limited amount of NAD+ in cell, NADH formed by this reaction must reoxidized to NAD+ to continue glycolysis) - Two major mechanisms for oxidizing NADH are: 1) NADH-linked conversion of pyruvate to lactate 2) oxidation of NADH via respiratory chain - Oxidation of CHO of GA-3P to COOH is coupled to attachment of Pi to carboxyl group - The high-energy P group at C-1 of 1,3-BPG conserves much of the free energy produced by oxidation of GA-3P - The energy of this high-energy P drives synthesis of ATP in the next reaction of glycolysis - 2,3 bisphosphoglycerate at high conc in RBCs G. Synthesis of 3-phosphoglycerate producing ATP - When 1,3-BPG is converted to 3- phosphoglycerate, the high-energy P group of 1,3-BPG is used to synthesize ATP from ADP - This reaction is catalyzed by phosphoglycerate kinase, - Because 2 molecules of 1,3-BPG are formed from each gluc molecule →this reaction replaces the 2 ATP consumed in formation of G-6-P & F-1,6-BP Note: this is an example of substrate level phospho., in which production of a high- energy P is coupled directly to oxidation of substrate, instead of resulting from oxidative phosph. via ETC. H. Shift of phosphate group from carbon 3 to 2 - by phosphoglycerte mutase is reversible I. Dehydration of 2-phosphoglycerate - Dehydration of 2-phosphoglycerate by enolase distributes the energy within 2- phosphoglycerate → formation of PEP, that contains a high-energy enol phosphate. - Reaction is reversible despite the high-energy nature of the product J. Formation of pyruvate producing ATP - Conversion of PEP to pyruvate is catalyzed by pyruvate kinase. 3rd irreversible rxn of glycolysis. - The equil of pyruvate kinase reaction favors formation of ATP - Note: this is another example of substrate level phosphorylation 1. Feed-forward regulation: - In liver, pyruvate kinase is activated by F-1,6- BP, the product of PFK reaction. This feed- forward regulation.This link the 2 kinase activities: increased PFK activity → elevated levels of F-1,6-BP → activates pyruvate kinas 2. Covalent modulation of pyruvate kinase (PK): - Phosphorylation by a cAMP-dependent protein kinase → inactivation of PK in liver - Low blood gluc → elevated glucagon → increases intracellular cAMP → PK phosphorylation & inactivation in liver only. - Therefore, PEP is unable to continue in glycolysis, instead enters gluconeogenesis. - Dephosphorylation of PK by a phosphoprotein phosphatase → PK reactivation Genetic defects of glycolytic enzymes - ~ 95% have deficiency in pyruvate kinase, & 4% phosphoglucose isomerase Pyruvate kinase deficiency: -Normal RBC lacks mitochondria → completely depends on glyolysis for production of ATP. -ATP required for metabolic needs of RBC, & to fuel pumps necessary for maintenance of bi-concave, flexible shape of cell, allows it to squeeze through narrow capillaries -PK defeciency→reduced rate of glycolysis →decreased ATP production → alterations in RBC memb → changes in shape of cell,& ultimately, to phagocytosis by cells of reticuloendothelial system, particularly spleen macrophages -Premature death & lysis of RBC → hemolytic anemia -Severity of disease depends both on degree of enz deficiency (generally 5-35% of normal levels), & on extent to which individual’s RBCs compensate by synthesizing increased levels of 2,3-BPG K. Reduction of pyruvate to lactate -Lactate dehydrogenase form lactate as final product of anaerobic glycolysis in euk cells as RBCs, lens & cornea of the eye, kidney medulla, testes, & leukocytes 1.Lactate formation in muscle: -In exercising skeletal muscle, production of NADH (by glycolysis & by TCA cycle) exceeds oxidative capacity of respiratory chain → high NADH/NAD+ ratio, favoring reduction of pyruvate to lactate. -During intense exercise, lactate accumulates in muscle →drop in intracellular pH→ cramps. -Then lactate diffuse into bloodstream, used by liver to make glucose 2. Lactate consumption: - Direction of LDH reaction depends on ratio of pyruvate to lactate & on ratio of NADH/NAD+ in cell. E.g., in liver & heart, ratio of NADH/NAD+ is lower than in exercising muscle. → oxidizes lactate (obtained from blood) to pyruvate - In liver, pyruvate →glucose by gluconeogenesis or oxidized in the TCA cycle - Heart muscle exclusively oxidizes lactate to CO2 & H2O via citric acid cycle 3. Lactic acidosis: - Elevated conc’s of lactate in plasma termed lactic acidosis occur when there is a collapse of the circulatory system, e.g., in MI, pulmonary embolism, & uncontrolled hemorrhage, or when an individual is in shock - Failure to bring adequate amounts of oxygen to the tissues → impaired oxidative phosphorylation & decreased ATP synthesis - To survive, cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the end-product Note: production of even meager amounts of ATP may be life-saving during the period required to re- establish adequate blood flow to the tissues - The excess oxygen required to recover from a period when the availability of oxygen has been inadequate is termed “oxygen debt”. - The oxygen debt is often related to patient morbidity or mortality. - In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of oxygen debt in patients and to monitor the patients’ recovery L. Energy yield from glycolysis - Despite production of some ATP during glycolysis, end-products, pyruvate or lactate, still contain most of energy originally contained in gluc. The TCA cycle is required to release that energy completely. 1. Anaerobic glycolysis: - Two molecules of ATP are generated for each molecule of gluc converted to 2 molecules of lactate. There is no net production or consumption of NADH. L. Energy yield from glycolysis -Glycolysis produce some ATP and end-products( pyruvate or lactate), still contain most of energy that was in gluc. TCA cycle will release that energy completely. 1. Anaerobic glycolysis: - Only two molecules of ATP are generated for each gluc molecule converted to 2 lactate molecules. No net production or consumption of NADH. 2. Aerobic glycolysis: Direct formation & consumption of ATP is the same as in anerobic glycolysis i.e., a net gain of 2 ATP -Two molecules of NADH are also produced per molecule of gluc - NADH by ETC, producing ~ 3 ATP for each NADH entering the chain (depending on the shuttle system) VI. Hormonal regulation of glycolysis -Short term (min. or hours): by allosteric activation or inhibition, or phospho/dephospho of rate-limiting enz’s -Slow (hours or days): hormones influence on amount enz protein synthesized. This can result in 10x to 20x fold increases in enz activity -Consumption of rich CHO meals or insulin administration → increase in amount of GK, PFK, & PK in liver -These changes reflect an increase in gene transcription→ result in increased enz synthesis. -Conversely, gene transcription & synthesis glucokinase, PFK, & PK are decreased when plasma glucagon is high & insulin is low, e.g., as seen in fasting or diabetes VII. Alternate fates of pyruvate A.Oxidative decarboxylation of pyruvate: - By pyruvate dehydrogenase complex important pathway in tissues with high oxidative capacity, e.g., cardiac muscle. - Irreversibly It converts pyruvate → acetyl CoA, major fuel for TCA & building block for F.A. B. Carboxylation to xaloacetate - By pyruvate caroxylase is a biotin-dependent reaction. Important reaction because it replenishes TCA cycle intermediates, & provides substrate for gluconeogenesis C- Reduction to ethanol (microorganisms) D- Decarboxylation by pyruvate decarboxylase occurs in yeast & certain m/o’s, but not in humans. The enz requires co-enzy. thiamine pyrophosphate & catalyzes a reaction similar to pyruvate dehydrogenase