Lecture 15 Ch 16 Glycolysis Glycolysis First phase Reactions 1 5 no ATP generated Second Phase Reactions 6 10 generation of ATP Feeder pathway to glycolysis other sugars can also enter the glycolytic pathway Regulation of glycolysis Glucose transporters Fate of pyruvate under anaerobic conditions In yeast Alcohol fermentation In other microorganisms and exercising muscle Lactic acid fermentation Hypoxia Inducible Factor 1 Metabolic pathways are compartmentalized in cells Phase 1 Phase 2 25 ATP Glycolysis in prokaryotes archaea eukaryotes Eukaryotic glycolysis occurs in the cytoplasm 2 1st Stage of Glycolysis Uses 2 molecules of ATP glucose but makes no ATP Traps and prepares glucose for following oxidation steps Glucose is converted to glyceraldehyde 3 phosphate GAP 5 steps 1 Phosphorylation of glucose 2 Isomerization 3 Second phosphorylation of Fructose 6P 4 Cleavage into two 3 carbon molecules 5 Isomerization of DHAP to GAP 3 Glycolysis Step 1 Phosphorylation Mg2 ATP H2O kJ mol glucose Pi glucose ATP ADP Pi G 6 P H2O G 6 P ADP G reaction 1 30 5 G reaction 2 14 kJ mol G overall 16 5 kJ mol 4 Hexokinase 5 Glucose is kept in the cell by phosphorylation to glucose 6phosphate 6 Glycolysis Step 2 Phosphoglucose Isomerase Glucose 6 phosphate G 6P Fructose 6 phosphate F 6P G6P is difficult to split into 3 carbon products F6P is easier to convert 7 Phosphoglucose Isomerase Mechanism 8 Glycolysis Reaction 3 Second Phosphorylation 9 Glycolysis Reactions 4 Cleavage of one 6 carbon to two 3 carbon molecules Fructose 1 6 bisphosphate ALDOLASE Readily reversible by Aldolase Both products need to be G3P for next step 10 Glycolysis Reactions 5 end of 1st phase of glycolysis 90 at equilibrium G3P is taken up by next step 11 Second Stage of Glycolysis The three carbon units are oxidized to pyruvate generating 4 molecules of ATP glucose and 2 NADH glucose Glyceraldehyde 3 Phosphaate G3P is converted to pyruvate 5 steps 6 Oxidation of G3P to 1 3BPG 7 Phosphorylation of ADP 8 Mutase 9 Dehydration by Enolase 10 Phosphorylation of ADP giving pyruvate 12 Glycolysis Reaction 6 G3P is oxidized to 1 3 BPG GAPDH Transfer Hydride from donor to NAD or from NADH to acceptor molecule Conversion of aldehyde to carboxylic acid before phosphorylation 2 step process 13 Uncoupled Oxidation and Phosphorylation Reactions aldehyde Carboxylic acid G 50 kJ mol 1 Requires Thioester intermediate G 56 kJ mol 1 Transfer Hydride from donor to NAD or from NADH to acceptor molecule Conversion of aldehyde to carboxylic acid before phosphorylation 2 step process 14 G3P Dehydrogenase Mechanism Nicotinamide adenine dinucletide Thiohemiacetal Oxidizing reagent Thioester Pi attacks carbonyl Thioester is higher energy than carboxylic acid Cannot resonate Less stable easier cleavage 15 Coupling of the two processes by one enzyme allows the conservation of energy released by oxidation G 6 3 kJ mol 16 Glycolysis Reaction 7 ATP producing reaction 2 per glucose Substrate level phosphorylation phosphate donor is kinase substrate Not a phosphatase because Pi is not generated as free molecule 2 ATP consumed in the 1st phase of glycolysis Reactions 1 and 3 are paid off in the this step of the 2nd phase of glycolysis 17 Phosphoglycerate Kinase Mechanism 18 Thermodynamically favorable Reaction 7 drives the less favorable Reaction 6 GAP G 6 3 kJ mol Step 6 1 3 BPG Preparative step for ATP production in the following step Compound with High Phosphoryl Group Transfer Potential G 49 6 kJ mol Step 7 ATP 30 5 kJ mol Actual ATP production 18 9 kJ mol 3 PG The formation of the high phosphoryl group transfer potential compound 1 3 BPG in step 6 is essential as a preparation for the ATP production in step 7 19 Glycolysis Reaction 8 Phosphoglycerate Mutase Note The term Mutase is applied to those enzymes that catalyze migration of functional groups from one position to another on the same substrate molecule 20 Glycolysis Reaction 9 Dehydration of 2 PG 21 Pyruvate kinase Ketone form G 31 7 kJ mol after removing the energy used to phosphorylate ADP 22 The net reaction for glycolysis Glucose 2Pi 2ADP 2NAD 2 pyruvate 2ATP 2NADH 2H 2H2O G 96 kJ mol 23 Entry points in glycolysis for galactose and fructose From Lactose From Sucrose hexokinase 2 pathways There are no dedicated pathways for other monosaccharides Other sugars must be converted to glycolysis intermediates 24 Fructose enters the glycolytic pathway in the liver through the fructose 1 phosphate pathway 25 Galactose enters the glycolytic pathway in the liver through the glucose 6 phosphate pathway Glucose 26 Glycosyltransferase Reaction UDP glucose phosphoglucomutase G 6P glycolysis 27 Regulation of Glycolysis 1 hexokinase 2 phosphofructokinase 3 pyruvate kinase Enzymes catalyzing irreversible reaction in metabolic pathways are potential control sites 28 1 Hexokinase versus Glucokinase Muscle contains Hexokinase I while liver contains Hexokinase IV Glucokinase This is to adapt to the different roles of muscle and liver Vmax 2 Km hexokinase 0 03 mM Km glucokinase 10 mM X normal blood sugar X Liver uses glucose left over from brain and muscle Hexokinase Liver uses glucose for glycogen storage G6P does NOT feed back to inhibit Glucokinase 29 Glucose 6 P is common to several metabolic pathways Hexokinase PFK 30 2 ofRegulation of Phosphofructokinase Regulation Allosteric Enzymes by feedback Mechanism 2 Regulation of Phosphofructose kianse Glucose HK G6P F6P PFK F1 6BP ATP Commitment Step Glycogen If G6P feeds into PFK then PFK activity can affect HK Eliminates repressor If PFK is off then G6P and F6P are at equilibrium G6P fed to Glycogen pathway Inhibition of PFK can inhibit KH 31 2 Regulation of Phosphofructokinase In muscles PFK ATP binds at 2nd site away from catalytic site When in excess Part of feedback Lowers affinity for F6P AMP reverses inhibition by ATP ADP is not used due to adenylate cyclase activity 2ADP 1ATP AMP 32 Allosteric Regulation of Phosphofructokinase High AMP Low AMP ATP is an allosteric inhibitor of PFK AMP competes off ATP inhibition of PFK 33 Regulation of Phosphofructokinase PFK PFK1 In muscles PFK In liver PFK is committed step in glycolysis G6P can be redirected to glycogen synthesis 34 Fructose 2 6 bisphosphate in Liver Blood Under hormonal I G regulation Increased F6P affinity 35 The activation of phosphofructokinase by fructose 2 6 bisphosphate Sigmoidal cooperative PFK is tetramer Allosteric inhibition 36 3 Regulatory mechanisms of pyruvate kinase In muscles M form and
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