Glycolysis PDF

Glycolysis Dr. Öğr Üyesi Gökhan BAĞCI Glycolysis - Overview ▪ One of best characterized pathways ▪ Characterized in the first half of 20th century ▪ Glucose ---------> 2 pyruvates + energy Strategy ✓ Add phosphoryl groups to glucose ✓ Convert phosphorylated intermediates into compounds with high...

Glycolysis Dr. Öğr Üyesi Gökhan BAĞCI Glycolysis - Overview ▪ One of best characterized pathways ▪ Characterized in the first half of 20th century ▪ Glucose ---------> 2 pyruvates + energy Strategy ✓ Add phosphoryl groups to glucose ✓ Convert phosphorylated intermediates into compounds with high phosphate group-transfer potentials ✓ Couple the subsequent hydrolysis of reactive substances to ATP synthesis Glucose + 2NAD+ + 2 ADP + 2Pi -->2NADH + 2 pyruvates + 2ATP + 2H2O + 4H+ Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway • Essentially all cells carry out glycolysis • Ten reactions - similar in most cells - but rates differ • Phases: • First phase converts glucose to two G-3-P • Second phase produces two pyruvates • Products are pyruvate, ATP and NADH • NADH must be recycled Glycolysis Three possible fates for pyruvate Mitochondrial oxidation 1 NADH --> ~3 ATP Reduction to lactate Decarboxylation to acetaldehyde Reduction to ethanol Enzymes of glycolysis Catalyzed reactions and properties Glucose Hexokinase, glucokinase Glucose-6-phosphate Phosphoglucoisomerase Fructose-6phosphate Phosphofructokinase Fructose-1,6biphosphate Dihydroxyacetone phosphate Triose phosphate isomerase Aldolase Glyceraldehyde-3phosphate First Phase of Glycolysis The first reaction - phosphorylation of glucose • Hexokinase or glucokinase • This is a priming reaction - ATP is consumed here in order to get more later • ATP makes the phosphorylation of glucose spontaneous • 1. Hexokinase: In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of • 2. Glucokinase: In liver parenchymal cells and β cells of the pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glucose. • In β cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion Hexokinase 1st step in glycolysis; G large, negative • Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell • Km for glucose is 0.1 mM; cell has 4 mM glucose • So hexokinase is normally active! • Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose • Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - Hexokinase • First step in glycolysis • Large negative deltaG • Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P • Corresponding reverse reaction (Gluconeogenesis) is catalyzed by a different enzyme (glucose-6phosphatase) Glucose Glucose-6-P dehydrogenase Glycogen Glucose-6-P Ribose-5-P + NADPH Fructose-6-P Glyceraldehyde-3-P Pyruvate ATP Nucleic acid synthesis Reducing power Rx 2: Phosphoglucoisomerase Rx 3: Phosphofructokinase • • • • • • • PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, neg delta G - means PFK is highly regulated ATP inhibits, AMP reverses inhibition Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high Glycolysis - Second Phase Metabolic energy produces 4 ATP • Net ATP yield for glycolysis is two ATP • Second phase involves two very high energy phosphate intermediates • . • 1,3 BPG • Phosphoenolpyruvate Glyceraldehyde-3phosphate Glyceraldehyde-3-phosphate dehydrogenase 1,3-biphosphoglycerate Phosphoglycerate kinase 3-phosphoglycerate Phosphoglycerate mutase 2-phosphoglycerate Enolase phosphoenolpyruvate Pyruvate kinase pyruvate Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP • These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis • Large, negative G - regulation! • Allosterically activated by AMP, Fructose-1,6bisP • Allosterically inhibited by ATP and acetyl-CoA The Fate of NADH and Pyruvate Aerobic or anaerobic?? • NADH is energy - two possible fates: • If O2 is available (in aerobic conditions), NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation • In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis The Fate of NADH and Pyruvate Aerobic or anaerobic?? • Pyruvate is also has energy - two possible fates: • Aerobic: citric acid cycle • Anaerobic: LDH makes lactate Pyruvate can go in three major directions after glycolysis • 1. Under aerobic conditions pyruvate is oxidized to Acetyl-CoA which can enter Citric acid (TCA) cycle. • 2. Under anaerobic conditions pyruvate can be reduced to ethanol (fermentation) or lactate • Under anaerobic conditions formation of ethanol and lactate is important in the oxidization NADH back to NAD+ • 3. Under aerobic conditions NADH is oxidized to NAD+ by the respiratory electron transport chain. Need to recycle NAD+ from NADH if gylcolysis is to continue under anaerobic conditions Lactate formation NADH NAD OH O O O H3C C H3C C O Pyruvate NADH NAD C H C O Lactate •In animals under anaerobic conditions pyruvate is converted to lactate by the enzyme lactate dehydrogenase •It is Important for the regeneration of NAD+ under anaerobic conditions. Cori Cycle • The circulatory systems of large animals are not efficient enough O2 transport to sustain long periods of muscular activity. •Anaerobic conditions lead to lactate accumulation and depletion of glycogen stores •Short period of intense activity must be followed by recovery period •Lactic acidosis causes blood pH to drop Alcohol Fermentation •Important for the regeneration of NAD+ under anaerobic conditions •Process common to microorganisms like yeast •Yields neutral end products (CO2 and ethanol) •CO2 generated important in baking where it makes dough rise and brewing where it carbonates beer. Control Points in Glycolysis Regulation of Hexose Transporters • Intra-cellular [glucose] are much lower than blood [glucose]. • Glucose imported into cells through a passive glucose transporter. • Elevated blood glucose and insulin levels leads to increased number of glucose transporters in muscle and adipose cell plasma membranes. Insulin Induced Exocytosis of Glucose Transporter 1. Regulation of Hexokinase • Glucose-6-phosphate is an allosteric inhibitor of hexokinase. • Levels of glucose-6-phosphate increase when down stream steps are inhibited. • This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis. • Hexokinase is not necessary the first regulatory step inhibited. 2. Regulation of PhosphoFructokinase (PFK-1) • PKF-1 has quaternary structure • Inhibited by ATP and Citrate • Activated by AMP and Fructose-2,6-bisphosphate • Regulation related to energy status of cell. PFK-1 regulation by adenosine nucleotides • ATP is substrate and inhibitor. • Binds to active site and allosteric site on PFK. • Binding of ATP to allosteric site increase Km for ATP • • • • AMP and ADP are allosteric activators of PFK. AMP relieves inhibition by ATP. ADP decreases Km for ATP Glucagon (a pancreatic hormone) produced in response to low blood glucose triggers cAMP signaling pathway that ultimately results in decreased glycolysis. Glucagon Regulation of PFK-1 in Liver •G-Protein mediated cAMP signaling pathway •Induces protein kinase A that activates phosphatase activity and inhibits kinase activity •Results in lower F-2,6-P levels decrease PFK-1 activity (less glycolysis) Regulation of PFK by Fructose-2,6-bisphosphate • Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotes •Formed from fructose-6-phosphate by PFK-2 •Degraded to fructose-6-phosphate by fructrose 2,6-bisphosphatase. •In mammals the 2 activities are on the same enzyme •PFK-2 inhibited by Pi and stimulated by citrate 3.Regulation of Pyruvate Kinase • Allosteric enzyme • Activated by Fructose-1,6-bisphosphate (example of feed-forward regulation) • Inhibited by ATP • When high fructose 1,6-bisphosphate present plot of [S] vs Vo goes from sigmoidal to hyperbolic. • Increasing ATP concentration increases Km for PEP. • In liver, PK also regulated by glucagon. Protein kinase A phosphorylates PK and decreases PK acitivty. Hormonal Regulation of Glycolysis Deregulation of Glycolysis in Cancer Cells • Glucose uptake and glycolysis is ten times faster in solid tumors than in non-cancerous tissues. • Tumor cells initally lack connection to blood supply so limited oxygen supply • Tumor cells have fewer mitochondrial, depend more on glycolysis for ATP • Increase levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved) Pasteur Effect • Under anaerobic conditions glycoysis proceeds at higher rates than during aerobic conditions • Slowing of glycolysis in presence of oxygen is the Pasteur Effect. • Cells sense changes in ATP supply and demand and modulate glycolysis Other Sugars can enter glycolysis ➢ Mannose can be phosphorylated to mannose-6-phosphate by hexokinase and then converted to fructose-6phosphate by phosphomannose isomerase. ➢ Fructose can be phosphorylated by fructokinase to form fructose-1 phosphate (F-1-P). F-1-P can then be converted to glyceraldehyde and DHAP by F-1-P aldolase. Triose kinase then converts glyceraldehyde to G-3-P. ➢ These reactions occur in Liver.