Glycolysis & PDH complex PDF

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GLYCOLYSIS & PDH COMPLEX 

Cell Respiration: - Oxidation of glucose to form ATP 1. Glycolysis 2. Pyruvate Dehydrogenase Reaction (PDH) 3. Citric Acid Cycle 4. Electron Transport chain 5. Oxidative phosphorylation 6. Reoxidation of cytoplasmic NADH -

Cellular Respiration Glycolysis: 6-carbon molecule (glucose) oxidation (* oxygen not required) two 3-carbon molecules of pyruvate (pyruvic acid)

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Fate of glucose in living systems - Glucose + 6O2 = 6CO2 + 6H2O δGo= -2840 kJ/mol - Glucose + 2NAD+ = 2Pyruvate + 2NADH + 2H+ dGo = -146 kJ/mol - 5.2% of total free energy that can be released by glucose is released in glycolysis.

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Significance of the Glycolysis Pathway - Principal route for glucose & other hexoses (fructose, galactose) metabolism. - Οnly pathway that is taking place in all the cells of the body. - Able to function under anaerobic condition. - In strenuous exercise, when muscle tissue lacks enough oxygen, anaerobic glycolysis forms the major source of energy for muscles. - However certain tissue like the heart muscle is adapted for aerobic performance, and hence possesses low glycolytic activity and survival rate under ischemic conditions - Glycolysis is the only source of energy in erythrocytes. - The glycolytic pathway may be considered as the preliminary step before complete oxidation. - The glycolytic pathway provides carbon skeletons for synthesis of non-essential amino acids as well as glycerol part of fat. - Most of the reactions of the glycolytic pathway are reversible  also used for gluconeogenesis. - All cells still utilize glycolysis.

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Glycolysis

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Occurs in the cytoplasm of virtually all the cells of the body Anaerobic: When inadequate O2 is available, e.g. during strenuous exercise, production of NADH exceeds the capacity of the electron transport chain  Pyruvate is converted to lactate by lactate dehydrogenase.

Net energy production= 2xATP

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Pathway has 10 reactions  can be considered as two distinctive phases

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1. Energy investment Sugar phosphates are synthesised at the expense of ATP  ADP The sugar is metabolically activated by phosphorylation 6C split into 2x3C sugar phosphates (triose phosphates) 2. Energy generation Further activation of triose phosphates to energy-rich compounds Reduced electron carriers are generated (NADH) The energy-rich compounds then transfer phosphate to ADP to form ATP  substrate-level phosphorylation= the generation of an energy-rich phosphate bond driven by the breakdown of a more energy-rich substrate

Importance of phosphorylated intermediates: - Possession of negative charge which inhibit their diffusion through membrane. - Conservation of free energy in high energy phosphate bond. - Facilitation of catalysis.

1) Hexokinase reaction: phosphorylation of hexoses - Enzyme present in most cell in liver  glucokinase (ISOENZYMES) - Requires Mg-ATP complex as substrate / uncomplexed ATP  competitive inhibitor - Enzyme catalyses the reaction by proximity effect  bringing 2 substrates in closer proximity - Enzyme undergoes conformational change upon binding with Glucose (inhibited allosterically by G6P).

Hexokinase Km low, high affinity for glu Non-specific  can phosphorylate any of hexoses Present in tissues, supply glucose to tissues even in low blood glucose concentration

Glucokinase Km high, low affinity for glu Specific  can phosphorylate only glucose Present in liver only, it removes glucose from blood after meal

Not affected by insulin Allosterically inhibited by glucose

Stimulated by glucose and insulin Not inhibited by glu-6-PO4

2) Phosphoglucose Isomerase or Phosphohexose Isomerase:

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Isomerization of Glucose-6-Phosphate  Fructose-6-phosphate = G6P F6P Reversible Requires Mg++ for its activity Specific for G6P and F6P

3) Phosphofructokinase-1 Reaction: - Transfer of phosphoryl group from ATP to C-1 of F6P  Fructose 1,6 bisphosphate. - IRREVERSIBLE  REGULATORY STEP - Phosphofructokinase-1 enzyme: one of the most complex regulatory enzymes, with various allosteric inhibitors and activators. - ATP is an allosteric inhibitor, and Fructose-2,6-biphosphate is an activator of this enzyme. - ADP and AMP also activate PFK-1 whereas citrate is an inhibitor.

4) Aldolase Reaction: Cleavage of Fructose 1,6 bisphosphate  glyceraldehyde-3-phosphate (an aldose) + dihydroxy acetone phosphate (a ketose). - Cleavage catalysed by Aldol condensation mechanism - Standard free energy  +ve  requires energy *** +ve  requires energy *** -ve  releases energy

5) Triose phosphate mutase reaction: - Dihydroxyacetone phosphate  glyceraldehyde-3-Phosphate - Reversible reaction - Catalysed by acid-base catalysts - Histidine-95 and Glutamate -165 of the enzyme are involved

6) Glyceraldehyde-3-phosphate dehydrogenase reaction (GAPDH): - GAP  Bisphosphoglycerate GADPH - Glyceraldehyde 3-phoshate + inorganic phosphate ↔ 1,3 Biphosphoglycerate - Inhibited by iodoacetate - First reaction of energy yielding step. Oxidation of aldehyde derives the formation of a high energy acyl phosphate derivative - An inorganic phosphate is incorporated in this reaction without any expense of ATP. - NAD+  cofactor, acts as an oxidizing agent. The free energy released in the oxidation reaction is used in the formation of acylphosphate.

7) Phosphoglycerate kinase Reaction:

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Transfer of phosphoryl group from-1,3-bisphosphoglycerate to ADP generating ATP. Inhibited by arsenate The name of this enzyme indicates its function for reverse reaction. Catalyses the formation by proximity effect. ADP-Mg bind on one domain and 1,3BPG binds on the other and a conformational change brings them together similar to hexokinase. 6th + 7th steps are coupled reaction generating ATP from the energy released by oxidation of 3-phosphoglyceraldehyde. This step generates ATP by SUBSTRATE-LEVEL PHOSPHORYLATION

8) Phosphoglycerate Mutase Reaction: - 3-phosphoglycerate  2-phosphoglycerate (2-PG) (conversion of...) - In active form, the phosphoglycerate mutase is phosphorylated at His-179. - Transfer of phosphoryl group form enzyme to 3-PG, generating enzyme bound 2,3biphosphoglycerate intermediate - In the last step of reaction the phosphoryl group from the C-3 of the intermediate is transferred to the enzyme and 2-PG is released. - In most cells 2,3-BPG is present in trace amount, but in erythrocytes it is present in significant amount  regulates oxygen affinity to haemoglobin.

9) Enolase Reaction: Dehydration of 2-phosphoglycerate (2-PG)  phosphoenolpyruvate (PEP) - Increases the standard free energy change of hydrolysis of phosphoanhydride bond - Mechanism: Rapid extraction of proton from C-2 position by a general base on enzyme - The second rate limiting step involves elimination of -OH group generating PEP - Inhibited by fluoride

10) Pyruvate Kinase Reaction: - Transfer of phosphoryl group from PEP to ADP generates ATP and Pyruvate. - Second substrate level phosphorylation reaction of glycolysis. - Enzyme couples the free energy of PEP hydrolysis to the synthesis of ATP - Requires Mg++ and K+

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Energetics and products of Glycolysis: - 1 molecule of Glucose: 1Gl+2ATP+2NAD++ 4ADP+ 4Pi = 2pyruvate+2NADH+4ATP+ 2ADP+ 2Pi - After balancing: 1Gl + 2NAD++ 2ADP + 2Pi = 2pyruvate+2ATP + 2NADH - The 2 NADH molecules are oxidized in mitochondria under aerobic condition and the free energy released is enough to synthesize 6 molecules of ATP by oxidative phosphorylation.

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Under the aerobic condition: pyruvate is catabolized further in mitochondria through pyruvate dehydrogenase and citric acid cycle where all the carbon atoms  oxidized to CO2 The free energy released is used in the synthesis of ATP, NADH and FADH2. Under anaerobic condition: Pyruvate  converted to Lactate in homolactic fermentation or in ethanol in alcoholic fermentation.

 GLYCOLYSIS:  Glucose + 2ADP + 2Pi + 2NAD+  2 pyruvate + 2ATP + 2NADH + 2H+

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Regulation of Glycolysis: - Two types of controls for metabolic reactions:  Substrate limited: When concentrations of reactant and products in the cell are near equilibrium, then it is the availability of substrate which decides the rate of reaction.  Enzyme-limited: When concentration of substrate and products are far away from the equilibrium, then it is activity of enzyme that decides the rate of reaction. These reactions are the one which control the flux of the overall pathway. - Three steps in glycolysis that have enzymes which regulate the flux of glycolysis. I. hexokinase (HK) II. phosphofructokinase (PFK) III. pyruvate kinase

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Next: - Oxidation of pyruvate - Generation of an activated 2C fragment  acetyl group of acetyl-CoA

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Pyruvate  Acetyl-CoA: - Pyruvate enters mitochondrial matrix undergoes oxidative decarboxylation - pyruvate + NAD+ + CoA  acetyl-CoA + NADH + CO2 - complicated sequence of reactions catalysed by an enzyme complex = pyruvate dehydrogenase complex = 3 enzymes + 5 coenzymes - virtually irreversible reaction - Then Acetyl-CoA enters the Citric Acid Cycle (central role in the cell metabolism, to oxidize organic metabolites)...