1-Glycolysis& Regulation[1].pdf

Glycolysis Regulation of Glycolysis OBJECTIVES Describe the overall purpose of glycolysis, its reactants and products, its cellular localization, and its tissue distribution Learn and understand the control(s) and control points of the glycolysis pathway. Three stages of catabolism Central Importanc...

Glycolysis Regulation of Glycolysis OBJECTIVES Describe the overall purpose of glycolysis, its reactants and products, its cellular localization, and its tissue distribution Learn and understand the control(s) and control points of the glycolysis pathway. Three stages of catabolism Central Importance of Glucose 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 – The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types For example: erythrocytes, brain, renal medulla 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 – provide ATP and metabolic intermediates Pentose Phosphate Pathway – Generates NADPH via oxidation of glucose – yield ribose 5-phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes The Glycolysis Pathway (from the Greek glykys, meaning “sweet,” and lysis, meaning “splitting”) Glycolysis is the sequence of reactions that metabolizes 1 molecule of glucose to 2 molecules of pyruvate with the concomitant net production of 2 molecules of ATP Carried out in the cytosol of cells Glycolysis – Anaerobic, evolved before significant O2 in atmosphere – Under anaerobic conditions, pyruvate can be converted to ethanol (alcoholic fermentation) or lactic acid (lactic acid fermentation) – Under aerobic conditions, pyruvate can be converted to CO2, producing much more ATP Glycolysis Has Two Phases Glycolysis: The Preparatory Phase Glycolysis involves "priming" steps which require energy, cleavage of the 6 carbon sugar into two three carbon molecules, and energy generation. Glycolysis: The Payoff Phase Step 1: Phosphorylation of Glucose Step 1: Phosphorylation of Glucose Rationale – Traps glucose inside the cell – Lowers intracellular glucose concentration to allow further uptake 1st priming reaction This process uses the energy of ATP Irreversable reaction Catalyzed by hexokinase (in liver& pancreas: glucokinase) Glukokinase vs Hexokinase Hexokinase: present in all cells of all organisms. Glucokinase: Hepatocytes and pancreatic-β cells They differ from each other in kinetic and regulatory properties Hexokinase has low Km and; therefore can efficiently use low levels of glucose. It is quickly saturated Glukokinase has a high Km; so it does not become saturated till very high levels of glucose are reached Glucose 6-phosphate is an important compound at the junction of several metabolic pathways (glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogenesis, and glycogenolysis). Step 2: Phosphohexose Isomerization Rationale: – C1 of fructose is easier to phosphorylate by PFK – Allows for symmetrical cleavage by aldolase Aldose-ketose isomerization – An aldose (glucose) can isomerize into a ketose (fructose) Step 3: Conversion of Fructose 6-Phosphate to Fructose 1,6 bis-Phosphate Rationale – 2nd Priming Phosphorylation – Further activation of glucose – Allows for 1 phosphate/3-carbon sugar after step 4 – fructose 1,6-bisphosphate is committed to become pyruvate and yield energy This process uses the energy of ATP Irreversible reaction Phosphofructokinase-1 is highly regulated – By ATP, fructose-2,6-bisphosphate, and other metabolites – Do not burn glucose if there is plenty of ATP Step 4: Cleavage of F-1,6-Bisphosphate Rationale – Cleavage of a six-carbon sugar into two three-carbon sugars: Dihydroxyacetone Phosphate (DHAP) Glyceraldehyde-3-Phosphate (GAP) – High-energy phosphate sugars – Glyceraldehyde-3-phosphate concentration kept low to pull reaction forward Step 5: Triose Phosphate Interconversion Rationale: – Allows glycolysis to proceed by one pathway Only GAP is the substrate for the next enzyme DHAP must be converted to GAP Completes preparatory phase – GAP concentration kept low to pull reaction forward Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1,3Bisphosphoglycerate Rationale: – Generation of a high-energy phosphate compound – Incorporates inorganic phosphate Oxidation of aldehyde with NAD+ gives NADH NAD+/NADH balance in the cell is important Step 7: 1st Production of ATP Rationale: Substrate-level phosphorylation to make ATP 1,3-bisphosphoglycerate is a highenergy compound – can donate the phosphate group to ADP to make ATP Step 8: Migration of the Phosphate Rationale: Be able to form high-energy phosphate compound Mutases catalyze the migration of functional groups Step 9: Dehydration of 2-PG to PEP Rationale – Generate a high-energy phosphate compound 2-Phosphoglycerate is not a good phosphate donor The loss of the water molecule from 2-phosphoglycerate causes a redistribution of energy within the molecule, greatly increasing the standard free energy of hydrolysis of the phosphoryl group Step 10: 2nd Production of ATP Rationale – Substrate-level phosphorylation to make ATP – Net production of 2 ATP/glucose Pyruvate kinase requires Mg++ or Mn++ for activity – Regulated by ATP, divalent metals, and other metabolites Chemical Logic of Glycolysis Summary of Glycolysis Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP 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 heavily regulated – Ensure proper use of nutrients – Ensure production of ATP only when needed Inhibitors of Glycolysis Iodoacetate and Arsenate: Inhibitors of Glyceraldehyde-3phosphate dehydrogenase Fluoride: Inhibitor of Enolase – is used to collect blood sample for plasma glucose determination TRANSPORT OF GLUCOSE INTO CELLS Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms: 1. Na+-independent, facilitated diffusion transport system (GLUT family; consists of 14 members) 2. Na+-monosaccharide cotransporter system (Nadependent glucose transporters) (SGLT family; sodiumglucose linked transporter ) in the epithelial cells of the intestine, renal tubules, and choroid plexus GLUT1 & GLUT3 – Present in almost all mammalian cells : responsible for basal glucose uptake – Km ~ 1 mM, serum glucose level 4-8 mM – Continually transport glucose into cells – GLUT-3 is the primary glucose transporter in neurons – GLUT-1 is abundant in erythrocytes and blood brain barrier GLUT2 – Present in liver, pancreatic β cells – Km ~ 15-20 mM : glucose rapidly enters liver cells only in times of plenty glucose level. Pancreas can thereby sense the glucose levels and accordingly adjusts insulin secretion – Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat GLUT4 – – – – – Insulin-dependent KM ~ 5mM Transports glucose into muscle & fat cells # of GLUT4 increases in presence of insulin Endurance training also increases # of GLUT4 GLUT5 – Present in small intestine – Functions primarily as fructose transporter Control of Glycolytic Pathway Glycolysis has 2 roles: 1. 2. degrades glucose to generate ATP provides building blocks for synthetic reactions How glycolysis is regulated? Enzymes catalyzing virtually irreversible reactions are potential regulation sites In glycolysis, these are hexokinase, pyruvate kinase, and phosphofructokinase These enzymes regulated by allosteric effectors (milliseconds), phosphorylation (seconds), & transcriptional control (hours) HORMONAL REGULATION OF GLYCOLYSIS Meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver Glucagon → diminishes the activity of these enzymes Elevation of blood glucose levels after a meal stimulates the pancreas to secrete insulin, which among its many actions induces synthesis of glucokinase by the liver. Control of Glycolytic Pathway (Muscle) Glycolysis is controlled in skeletal muscle – Primary function of glycolysis in muscle is to provide ATP for muscle contraction (keep energy charge high, with high ATP: AMP ratio) How does each enzyme respond to changes in [ATP] and [AMP]? Regulation of glycolysis in muscle Control of Glycolytic Pathway (Muscle) Hexokinase: How does hexokinase respond to changes in [ATP] and [AMP]? – Catalyzes Glucose → Glucose 6-phosphate (G6P) – is inhibited by high [G6P], means cell doesn’t require glucose for energy or synthesis of glycogen – When phosphofructokinase is inactive, [F6P] rises. In turn, the level of [G6P] rises. – High [G6P] means phosphofructokinase (PFK) is inactive Hence, the inhibition of phosphofructokinase leads to the inhibition of hexokinase Phosphofructokinase (PFK) – is the most important control site in the mammalian glycolytic pathway – allosterically inhibited by high levels of ATP, activated by AMP PFK also inhibited by deacrease in pH – when muscle is functioning anaerobically, producing excessive quantities of lactic acid – inhibitory effect protects the muscle from damage Control of Glycolytic Pathway (Muscle) Pyruvate Kinase How does pyruvate kinase (PK) respond to changes in [ATP] and [AMP]? – Products of PK are ATP and pyruvate – High [ATP] means energy charge high, and it allosterically inhibits PK – Alanine also inhibits PK (pyruvate → alanine), signals an abundance of building blocks Control of Glycolytic Pathway (Liver) Liver maintains blood glucose levels – Stores glucose as glycogen when glucose is plentiful – Releases glucose when supplies are low – Uses glucose to make reducing power for biosynthesis – Uses glucose as a precursor to synthesize other biomolecules Control of Glycolytic Pathway (Liver) Glucokinase – Liver has an isozyme, glucokinase that is NOT inhibited by G-6P – phosphorylates glucose only when glucose is abundant – provides glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acids Glucokinase activity: – indirectly inhibited by fructose 6-phosphate – indirectly stimulated by glucose – GKRP in the liver regulates the activity of glucokinase In the presence of F6-P, glucokinase is translocated into the nucleus and binds tightly to the regulatory protein, thus rendering the enzyme inactive Regulation of glucokinase activity by GKRP When glucose levels increase, glucokinase is released from the regulatory protein, and the enzyme reenters the cytosol where it phosphorylates glucose G6-P Control of Glycolytic Pathway (Liver) Phosphofructokinase (PFK) – Regulation by ATP same as in muscle cells – Low pH is not a metabolic signal for liver, because in liver lactic acid is converted→ glucose – Concentration of building blocks regulates PFK – In liver, PFK is inhibited by citrate, an early intermediate in the citric acid cycle Control of Glycolytic Pathway (Liver) Fructose 2,6-bisphosphate (F-2,6-BP) activates PFK – When [glucose] is high, [F6P] rises – Excess F6P speeds up synthesis of F2,6-BP – F-2,6-BP; is made by the action of a second PFK-2, using fructose 6phosphate and ATP as substrates. – Binding of F-2,6-BP increases PFK’s affinity for F6P and decreases ATP’s ability to inhibit PFK PFK-2 feedforward stimulation Control of Pyruvate Kinase (PK) – Allosteric regulation of liver and muscle PK is similar but differ in covalent modification – Liver form is controlled by reversible phosphorylation, muscle form not When the blood-glucose level is low : the glucagon-triggered cyclic AMP → phosphorylation of pyruvate kinase → diminishes the activity. Elevation of blood glucose levels after a meal stimulates the pancreas to secrete insulin, which among its many actions induces synthesis of glucokinase by the liver.