Lecture 03 Glycolysis.pdf

Glycolysis  Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway Essentially all cells carry out glycolysis Ten reactions - same in all cells - but rates differ Two phases: – First phase converts glucose to two G-3-P – Second phase produces two pyruvates...

Glycolysis  Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway Essentially all cells carry out glycolysis Ten reactions - same in all cells - but rates differ Two phases: – First phase converts glucose to two G-3-P – Second phase produces two pyruvates Products are pyruvate, ATP and NADH Three possible fates for pyruvate  Glycolysis https://www.youtube.com/watch?v=6ltUkb5x1_0  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  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 - but is not the most important site of regulation of glycolysis - Why? What is the difference between hexokinase and glucokinase? Glucokinase and hexokinase both do catalyze same reaction Hexokinase is present in all the cells and glucokinase is present in liver (and pancreas) cells. Normal cells do glycolysis to get the energy so they have hexokinase which have a lower Km which results in higher affinity of the enzyme towards glucose to do faster and efficient glycolysis. Liver cells first convert glucose in glycogen. To make glycogen efficiently liver cells have to slow down glycolysis so they have an enzyme glucokinase (higher value of Km) results into slower and lesser glycolysis.  Rx 2: Phosphoglucoisomerase Glucose-6-P to Fructose-6-P Why does this reaction occur?? – next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH – isomerization activates C-3 for cleavage in aldolase reaction  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 High [ATP] inhibits PFK, decreasing the enzyme’s affinity for fructose-6-phosphate. Fructose-2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate Fructose-2,6-bisphosphate decreases the inhibition of phosphofructokinase due to ATP  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  Rx 6: Gly-3-Dehydrogenase Gly-3P is oxidized to 1,3-BPG Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH Mechanism involves covalent catalysis and a nicotinamide coenzyme  Rx 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate This is referred to as "substrate-level phosphorylation" The other kind of phosphorylation, oxidative phosphorylation, is driven energetically by the transport of electrons from appropriate coenzymes and substrates to oxygen  Rx 9: Enolase 2-P-Gly to PEP Overall G is 1.8 kJ/mol How can such a reaction create a PEP? "Energy content" of 2-PG and PEP are similar Enolase just rearranges to a form from which more energy can be released in hydrolysis Rx 9: Enolase  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, F-1,6-bisP Allosterically inhibited by ATP and acetyl- CoA  The conversion of phosphoenolpyruvate (PEP) to pyruvate in two steps: phosphoryl transfer followed by an enol-keto tautomerization. The tautomerization is spontaneous (ΔG°’- 35–40 kJ/mol) and accounts for much of the free energy change for PEP hydrolysis  The Fate of NADH and Pyruvate Aerobic or anaerobic?? NADH is energy - two possible fates: – If O2 is available, 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 Py Aerobic or anaerobic?? Pyruvate is also energy - two possible fates: – aerobic: citric acid cycle – anaerobic: alcoholic fermentation, lactic acid fermentation Energetics of Glycolysis  Energetics of Glycolysis The elegant evidence of regulation! Standard state G values are scattered: + and - G in cells is revealing: – Most values near zero – 3 of 10 Rxns have large, negative  G Large negative  G Rxns are sites of regulation!  Other Substrates for Glycolysis Fructose, mannose and galactose Fructose and mannose are routed into glycolysis by fairly conventional means. Fructose two ways to enter – Liver – kidney and in muscle tissues  Fructose entry to glycolysis: In the liver fructose is phosphorylated at C-1 by the enzyme fructokinase. Fructose-1-phosphate aldolase cleaves fructose-1-P to produce dihydroxyacetone phosphate and D- glyceraldehyde Dihydroxyacetone phosphate is an intermediate in glycolysis. D-Glyceraldehyde can be phosphorylated by triose kinase in the presence of ATP to form D- glyceraldehyde-3-phosphate, another glycolytic intermediate.  Fructose entry to glycolysis: kidney and in muscle tissues fructose is readily phosphorylated by hexokinase, which, can utilize several different hexose substrates. The free energy of hydrolysis of ATP drives the reaction forward. Fructose-6-phosphate generated in this way enters the glycolytic pathway directly in step 3, the second priming reaction. This is the principal means for channeling fructose into glycolysis in adipose tissue, which contains high levels of fructose.  Galactose metabolism Normally, the body breaks down lactose into – galactose and then into – glucose (a sugar used for energy). People with galactosemia are missing (galactose-1- phosphate uridyl transferase), GALK1 or GALE, which normally converts galactose into glucose. Without this enzymes, harmful amounts of galactose build up in the blood. Lactose metabolism  Most programs detect galactosaemia by measuring ‘galactose metabolites’ (galactose and galactose-1- phosphate using enzymatic assays) Measurement of GALT activity is usually used as a second-tier test to help distinguish between the different forms of galactosaemia. Genetic test can be done on adults to find out whether they have an increased chance of having a child with the disease. Glycerol Can Also Enter Glycolysis  Fermentation of Glucose and Other Sugars Glucose Pyruvate CO2 Formate Lactate Oxaloacetate 2H Acetyl-CoA Malate Acrylate Fumarate Acetoacetyl CoA Succinate Methane Acetate Butyrate Propionate Succinyl CoA Propionyl CoA Methylmalonyl CoA Co Vit B12