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Metabolism – Intro to Metabolism CH339K. Going back to the early lectures.

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Presentation on theme: "Metabolism – Intro to Metabolism CH339K. Going back to the early lectures."— Presentation transcript:

1 Metabolism – Intro to Metabolism CH339K

2 Going back to the early lectures


4 Why the big G o for Hydrolyzing Phosphoanhydrides? Electrostatic repulsion betwixt negative charges Resonance stabilization of products pH effects

5 pH Effects – G o vs. G o G in kcal/mol) WOW!

6 Cellular Gs are not G o s G o for hydrolysis of ATP is about -31 kJ/mol Cellular conditions are not standard, however: In a human erythrocyte, [ATP]2.25 mM, [ADP] 0.25 mM, [PO 4 ] 1.65 mM

7 Unfavorable Reactions can be Subsidized with Favorable Ones


9 Activation with ATP - luciferin Excited state of oxyluciferin forms and decays

10 For those who prefer more detail Excerpted from Baldwin, T. (1996) Structure 4: 223 – 228,

11 Tobacco seedling w/ cloned luciferase Southeast Asian firefly tree Just because its cool…

12 New Zealand glowworm (Arachnocampa) cave Firefly squid (Watasenia scintillans ) of Toyama Bay, Japan

13 Hydrolysis of Thioesters can also provide a lot of free energy

14 Acetyl Coenzyme A

15 Sample G o Hydrolysis

16 Phosphate Transfer Potential is a fancy-schmancy term for – G o

17 1.10 V Electrochemistry in review One beaker w/ ZnSO 4 and a Zn electrode One beaker w/ CuSO 4 and a Cu electrode Zinc gets oxidized and the electrode slowly vanishes Copper gets reduced and the electrode gets fatter

18 Standard Hydrogen Electrode

19 Redox Table Higher the SRP, the better the oxidant Lower the SRP, the better the reductant Any substance can oxidize any substance below it in the table. The number of reactants involved doesnt change the reduction potential i.e. if a reaction involves 2 NAD +, the SRP is still V

20 1.10 V Electrochemistry in review Zinc gets oxidized Copper gets reduced What determines who gets oxidized?

21 E o and K eq For an actual half reaction aA + ne - aA - For an actual redox reaction: A +n + ne- A B B +n + ne- A +n + B A + B +n and (Analagous to the relation between G and G o )

22 E o and K eq (cont.) At equilibrium, the two are equal: Combining: Or Or (rearranging) Dr. Ready gets to the Point!

23 E o and G o So: But we already know: Therefore: Another Point!

24 NAD + Reduction (Nicotinamide Adenine Dinucleotide) NAD + is a common redox cofactor in biochemistry

25 Coenzyme Q Coenzyme Q is another electron carrier in the cell

26 An Example: What is G o for the Oxidation of NADH by Ubiquinone?

27 Cigarettes Vitamins

28 Organic Healthy LD – 1.0 mg / kg Vomiting and nausea, diarrhea, Headaches, Difficulty breathing, Pallor, Sweating, Palpitations, Lisps, Stomach pains/cramps, Seizures, Weakness, Drooling, and - of course - Death

29 Flavins

30 Energy (ATP) Parts (amino acids, etc.) Reducing Power (NADH, NADPH) Catabolism (Oxidation) Anabolism (Reduction)

31 Fates of Glucose

32 Catabolism of Glucose C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O G o = kJ/mol It takes 31 kJ/mol to make an ATP. Enough energy is available for making ~90 (theoretically)

33 An aside on diets Glucose (a carb), mol. wt. = 180 g/mol kJ/mol = -686 kcal/mol -686 kcal/mol / 180 g/mol = 3.8 kcal/g Palmitic Acid (a fatty acid) mol. wt. = 256 g/mol kJ/mol = kcal/mol kcal/mol / 256 g/mol = 9.3 kcal/g Alanine (an amino acid) mol. wt. = 88 g/mol kJ/mol = -310 kcal/mol -310 kcal/mol / 88 g/mol = 3.5 kcal/g

34 An aside on diets (cont.) From Fat: 1 gram = 9 calories Protein: 1 gram = 4 calories Carbohydrates: 1 gram = 4 calories The diet values come from the G o for oxidizing the various biomolecules.

35 Catabolism of Glucose

36 Interconversion of C 6 Sugars Glucose-1-Phosphate Glucose-6-Phosphate Fructose-6-Phosphate Glycogen GlucoseAmino Sugars Nucleotides Fatty Acids -7.3 kJ/mol -0.4 kJ/mol Catabolism


38 Glucose Catabolism Part 1: Glycolysis Aka Embden-Meyerhof pathway Worked out in the 1930s Partially oxidizes glucose Uses no O 2 Takes place in cytoplasm

39 Interconversion of C 6 Sugars (Again) Glucose-1-Phosphate Glucose-6-Phosphate Fructose-6-Phosphate Glycogen GlucoseAmino Sugars Nucleotides Fatty Acids -7.3 kJ/mol -0.4 kJ/mol Catabolism Phosphoglucomutase Phosphohexose isomerase

40 Dont Eat the Toothpaste! Phosphoglucomutase contains a PO 4 -2 group attached to residue D8. Fluoride has a number of toxic effects One of them is the removal of the phosphate from phosphoglucomutase No phosphate = no activity No activity = cant utilize glycogen



43 Glycolysis - Energetics

44 Phosphohexose Isomerase

45 Aldolase

46 Aldolase Reaction The standard free energy, G o,for the aldolase reaction is very unfavorable (~ +25 kJ/mol) Under cellular conditions, the real free energy, G, is favorable (~ -6 kJ/mol) [G-3P] is maintained well below the equilibrium level by being processed through the glycolytic pathway

47 Triose Phosphate Isomerase

48 Gyceraldehyde-3-P Dehydrogenase

49 Phosphoglyceromutase H8 in human erythrocyte PGM

50 Overall Reaction The overall reaction of glycolysis is: Glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP + 2 H2O + 4 H + There is a net gain of 2 ATP per glucose molecule As glucose is oxidized, two NAD+ are reduced to 2 NADH

51 When two things look alike… …there can be a problem.

52 Arsenate Poisoning (in part) G3P Dehydrogenase will happily use arsenate as a substrate. 1-Arseno-3-phosphoglycerate decomposes spontaneously without production of ATP. Primary poisoning effect is on a different part of catabolism

53 Why does arsenic poisoning ever come up? Chromated copper arsenate was the primary agent for pressure treated wood in the USA until 2003 Mono- and disodium methyl arsenate are used as agricultural insecticides Arsphenamine was one of the first treatments for syphilis Arsenic trioxide is an approved treatment for promyelocytic leukemia Lewisite is an old-fashioned CBW blister and lung agent Coppers acetoarsenite is Paris green, a pigment used by artists, some of whom had the habit of licking their brushes Scheeles Green (copper arsenite) was used as a coloring agent for candy in the 19 th century

54 Relation to Hb Oxygenation

55 Glycolysis – Genetic Defects

56 Antitrypanosomals Remember these guys? Chagas Disease African Sleeping Sickness Nagana Leishmaniasis (Baghdad Boil) Afflict hundreds of millions Nagana responsible for the popularity of cannibalism in the African fly belt. Leishmaniasis is now endemic in Texas

57 Antitrypanosomals Trypanosomes have unusual glycolysis enzymes First 7 steps carried out in glycosomes Enzymes are quite different in structure and sequence from mammalian enzymes Good drug targets

58 Antitrypanosomals Model of L. mexicana glyceraldehyde-3-phosphate dehydrogenase complexed with N6-(1- naphthylmethyl)-2¢-deoxy-2¢- (3-methoxybenzamido)-adenosine.

59 Antitrypanosomals Binding mode of 2-amino-N6-(p-hydroxyphenethyl)adenosine to T. brucei phosphoglycerate kinase.

60 Energetics of Glycolysis G o values are scattered: + and - G in cells is revealing: Most values near zero 3 of 10 Rxns have large, negative G (i.e. irreversible) Large negative G Rxns are sites of regulation!

61 Glycolysis - Regulation

62 Hexokinase regulation Hexokinase – muscle –Km for glucose is 0.1 mM; cell has 4 mm glucose –So hexokinase is normally active! –Allosterically inhibited by (product) glucose-6-P (product inhibition) Glucokinase – liver, pancreas –Km glucose 8 mM (144 mg/dl – above normal) –Cooperative – nH 1.7 –No product inhibition –Only turns on when cell is rich in glucose –Shifts hepatocytes from fasting to fed metabolic states, encouraging glycogen synthesis and glycolysis –Acts as signal in pancreas to release insulin

63 Hexokinase vs. Glucokinase

64 PFK PFK is a tetrameric protein that exists in two conformational states - R and T (i.e. cooperative) High concentrations of ATP shift the T R equilibrium in favor of the T state decreasing PFKs affinity for F6P AMP, ADP and Fructose 2,6 Bisphosphate acts to relieve inhibition by ATP

65 Fates of Pyruvate Pyruvate AcetylCoAEthanolLactate (Yeast, no O 2 )(Critters, no O 2 )(Aerobic) In the absence of O2, no further oxidation occurs. NADH builds up, and NAD+ has to be regenerated to continue glycolysis

66 NADH Regeneration

67 Yeasties: Alcohol Dehydrogenase Pyruvate Decarboxylase Alcohol Dehydrogenase

68 Critters: Lactate Dehydrogenase Lactate Dehydrogenase

69 Glucose Catabolism Part 2 Pyruvate Dehydrogenase Huge multienzyme complex –4.6 Mdaltons in E. Coli ( ) –9 Mdaltons in mammals ( ) 3 separate enzyme functions create overall reaction Pyruvate + NAD + + HSCoA CO 2 + Acetyl CoA + NADH This is where we actually lose our first carbon(s) from glucose

70 Pyruvate Dehydrogenase - Reaction

71 PDH - Subunits SubunitEnzyme FunctionCofactorNumber In Prokaryotes Number In Eukaryotes (or E1) Pyruvate Dehydrogenase Thiamine Pyrophosphate 2430 (or E2) Dihydrolipoamide Transacetylase Lipoic Acid2460 (or E3) Dihydrolipoamide Dehydrogenase Flavin Adenine Dinucleotide 12

72 PDH - Structure

73 PDH - Schematic

74 E1 – Pyruvate Dehydrogenase Proper In E. coli, E1 is a dimer of two similar subunits In mammals, E1 is an 2 2 tetramer. Each E1 contains 2 active sites Each active site contains a thiamine pyrophosphate cofactor. TPP is ligated to a metal ion and is H-bonded to several amino acids

75 Pyruvate Dehydrogenase – Thiamine Pyrophosphate Hydrogen is Acidic

76 Pyruvate Dehydrogenase

77 E2 – Dihydrolipoamide Transacetylase Lipoic Acid In enzyme, Lipoic Acid is attached to a lysine Disulfide is at end of very long floppy arm Can bounce back and forth between PDC and DHLD on surface


79 Coenzyme A Thioesters are activated compounds Coenzyme A is a common activator Warhead of CoA is the thiol –Hence, abbreviated HS-CoA

80 Dihydrolipoamide Transacetylase Lipoamide is reduced Accepts acyl unit from PDC / Thiamine PP Transfers to CoA

81 FAD

82 E3 - Dihydrolipoamide Dehydrogenase

83 PDH - Overall

84 Organic arsenicals are potent inhibitors of lipoamide- containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with vicinal dithiols such as the functional group of lipoamide.

85 Product inhibition by NADH & acetyl CoA: NADH competes with NAD + for binding to E 3. Acetyl CoA competes with CoA for binding to E 2. PDH Regulation

86 Regulation by E 1 phosphorylation/dephosphorylation: Specific regulatory Kinases & Phosphatases associated with Pyruvate Dehydrogenase in the mitochondrial matrix: Pyruvate Dehydrogenase Kinases catalyze phosphorylation of serine residues of E 1, inhibiting the complex. Pyruvate Dehydrogenase Phosphatases reverse this inhibition. Pyruvate Dehydrogenase Kinases are activated by NADH & acetyl-CoA, providing another way the 2 major products of Pyruvate Dehydrogenase reaction inhibit the complex. PDH - Regulation


88 During starvation: Pyruvate Dehydrogenase Kinase increases in amount in most tissues, including skeletal muscle, via increased gene transcription. Under the same conditions, the amount of Pyruvate Dehydrogenase Phosphatase decreases. The resulting inhibition of Pyruvate Dehydrogenase prevents muscle and other tissues from catabolizing glucose & gluconeogenesis precursors. Metabolism shifts toward fat utilization. Muscle protein breakdown to supply gluconeogenesis precursors is minimized. Available glucose is spared for use by the brain.


90 Overall Reaction Per glucose that entered glycolysis: Thus, at the end of the cycle, we will have converted our glucose completely to CO 2. We still wont have used any oxygen or made any water.

91 Location Also known as citric acid cycle, tricarboxylic acid cycle Krebs takes place in the mitochondrial matrix One enzyme is an integral membrane protein of the IMM


93 At Equilibrium Citrate91% Cis-Aconitate3% Isocitrate6%

94 Stereospecificity of Aconitase Recognized back in 1956 that aconitase dehydrates across a particular bond in citrate (England et al (1957) J. Biol. Chem. 226: 1047) Citrate is not chiral Multipoint binding allows stereospecificity in a nonchiral compound

95 An Aconitase Inhibitor Sodium Fluoroacetate is a fairly potent toxin (2-10 mg/kg) Brand name 1080 Incoporated into fluoroacetylCoA, then into fluorocitrate Fluorocitrate is a powerful competitive inhibitor of aconitase

96 Coyote Control by 1080

97 1)Oxidation: NAD + oxidizes the hydroxyl carbon of isocitrate 2)Decarboxylation: A Mn +2 bound to the enzyme stabilizes the intermediate 3)Protonation: Reforms the carbonyl to generate product 4)General Principle: NAD + is usually the electron recipient when oxidizing at a hydroxyl Isocitrate Dehydrogenase G o = kJ/mol

98 Weve now lost 2 CO 2 in Krebs + 1 in PDH – glucose is gone. The two carbons weve lost are not the same ones we brought in.

99 Substrate level phosphorylation Plants make ATP directly Critters make GTP, then exchange phosphate to ATP

100 Succinyl CoA Synthetase Rxn 1.CoA is displaced by an Orthophosphate 2.The phosphate group is transferred to a Histidine residue on the enzyme 3.Succinate leaves as a product 4.The enzyme is dephosphorylated, passing PO 4 -3 to a nucleotide diphosphate

101 General Principle: FAD is the preferred cofactor for oxidizing a carbon-carbon bond. Succinate Dehydrogenase is an integral membrane protein

102 Water attacks the double bond in a 2-step process.



105 1.) Citrate Synthase6.) Succinate Dehydrogenase 2.) Aconitase7.) Fumarase 3.) Isocitrate Dehydrogenase8.) Malate Dehydrogenase 4.) α-Ketoglutarate Dehydrogenase9.) Overall reaction 5.) Succinyl-CoA Synthetase G o G

106 Reaction Enzyme G°' (kJ/mol) 1Citrate synthase Aconitase+6.3 3Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase complex Succinyl-CoA synthetase-2.9 6Succinate dehydrogenase0.0 7Fumerase-3.8 8Malate dehydrogenase+29.7 Krebs Cycle Energetics

107 The citric acid is regulated by three simple mechanisms. 1. Substrate availability 2. Product inhibition 3. Competitive feedback inhibition.

108 The Krebs cycle is amphibolic – intermediates are also used to make stuff.

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