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Metabolism – Intro to Metabolism

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1 Metabolism – Intro to Metabolism
CH339K

2 Going back to the early lectures

3

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

5 pH Effects – DGo vs. DGo’ (DG in kcal/mol) WOW!

6 Cellular DGs are not DGo’ s
DGo’ 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, [PO4] ≈1.65 mM

7 Unfavorable Reactions can be Subsidized with Favorable Ones

8

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 Just because it’s cool…
Tobacco seedling w/ cloned luciferase Southeast Asian firefly tree

12 Just because it’s cool…
Firefly squid (Watasenia scintillans ) of Toyama Bay, Japan New Zealand glowworm (Arachnocampa) cave

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

14 Acetyl Coenzyme A

15 Sample DGo’Hydrolysis

16 “Phosphate Transfer Potential” is a fancy-schmancy term for –DGo’

17 Electrochemistry in review
One beaker w/ ZnSO4 and a Zn electrode One beaker w/ CuSO4 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 doesn’t change the reduction potential i.e. if a reaction involves 2 NAD+, the SRP is still V

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

21 DEo and Keq 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 DG and DGo’)

22 DEo and Keq (cont.) At equilibrium, the two are equal: Combining: Or
Or (rearranging) Dr. Ready gets to the Point!

23 DEo and DGo 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 DGo’ for the Oxidation of NADH by Ubiquinone?

27 Cigarettes ≠ Vitamins

28 “Organic” ≠ “Healthy” LD50 0.5 – 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 Metabolism Energy (ATP) Parts (amino acids, etc.)
Reducing Power (NADH, NADPH) Catabolism (Oxidation) Anabolism (Reduction)

31 Fates of Glucose

32 C6H12O6 + 6O2 → 6CO2 + 6H2O DGo’ = -2870 kJ/mol Catabolism of Glucose
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
-2870 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 -9959 kJ/mol = kcal/mol -2380 kcal/mol / 256 g/mol = 9.3 kcal/g Alanine (an amino acid) mol. wt. = 88 g/mol -1297 kJ/mol = -310 kcal/mol -310 kcal/mol / 88 g/mol = 3.5 kcal/g

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

35 Catabolism of Glucose

36 Interconversion of C6 Sugars
Glycogen Glucose-1-Phosphate -7.3 kJ/mol Glucose Glucose-6-Phosphate Amino Sugars -0.4 kJ/mol Nucleotides Fatty Acids Fructose-6-Phosphate Catabolism

37 STOP HERE FOR INTRO LECTURE

38 Glucose Catabolism Part 1: Glycolysis
Aka Embden-Meyerhof pathway Worked out in the 1930’s Partially oxidizes glucose Uses no O2 Takes place in cytoplasm

39 Interconversion of C6 Sugars (Again)
Glycogen Glucose-1-Phosphate Phosphoglucomutase -7.3 kJ/mol Glucose Glucose-6-Phosphate Amino Sugars -0.4 kJ/mol Phosphohexose isomerase Nucleotides Fatty Acids Fructose-6-Phosphate Catabolism

40 Don’t Eat the Toothpaste!
Phosphoglucomutase contains a PO4-2 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 = can’t utilize glycogen

41

42

43 Glycolysis - Energetics

44 Phosphohexose Isomerase

45 Aldolase

46 Aldolase Reaction The standard free energy , DGo,for the aldolase reaction is very unfavorable (~ +25 kJ/mol) Under cellular conditions, the real free energy, DG, 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 Scheele’s Green (copper arsenite) was used as a coloring agent for candy in the 19th century

54 Relation to Hb Oxygenation

55 Glycolysis – Genetic Defects

56 Antitrypanosomals 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 Remember these guys?

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
DGo values are scattered: + and - DG in cells is revealing: Most values near zero 3 of 10 Rxns have large, negative DG (i.e. irreversible) Large negative DG 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 PFK’s affinity for F6P AMP, ADP and Fructose 2,6 Bisphosphate acts to relieve inhibition by ATP

65 Fates of Pyruvate Pyruvate Ethanol Lactate AcetylCoA
(Yeast, no O2) (Critters, no O2) (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

68 Critters: Lactate Dehydrogenase

69 Glucose Catabolism Part 2 Pyruvate Dehydrogenase
Huge multienzyme complex 4.6 Mdaltons in E. Coli (a24b24g12) 9 Mdaltons in mammals (a60b60g24) 3 separate enzyme functions create overall reaction Pyruvate + NAD+ + HSCoA  CO2 + Acetyl CoA + NADH This is where we actually lose our first carbon(s) from glucose

70 Pyruvate Dehydrogenase - Reaction

71 PDH - Subunits Subunit Enzyme Function Cofactor Number In Prokaryotes
Eukaryotes a (or E1) Pyruvate Dehydrogenase Thiamine Pyrophosphate 24 30 b (or E2) Dihydrolipoamide Transacetylase Lipoic Acid 60 g (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 a2b2 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

78

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 PDH Regulation NADH competes with NAD+ for binding to E3.
Product inhibition by NADH & acetyl CoA: NADH competes with NAD+ for binding to E3. Acetyl CoA competes with CoA for binding to E2.

86 PDH - Regulation Regulation by E1 phosphorylation/dephosphorylation: Specific regulatory Kinases & Phosphatases associated with Pyruvate Dehydrogenase in the mitochondrial matrix: Pyruvate Dehydrogenase Kinases catalyze phosphorylation of serine residues of E1, 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.

87

88 Metabolism shifts toward fat utilization.
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.

89 The Krebs CYCLE

90 Overall Reaction Per glucose that entered glycolysis:
Thus, at the end of the cycle, we will have converted our glucose completely to CO2. We still won’t 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

92

93 At Equilibrium Citrate 91% Cis-Aconitate 3% Isocitrate 6%

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

98 We’ve now lost 2 CO2 in Krebs + 1 in PDH – glucose is gone.
The two carbons we’ve 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
CoA is displaced by an Orthophosphate The phosphate group is transferred to a Histidine residue on the enzyme Succinate leaves as a product The enzyme is dephosphorylated, passing PO4-3 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.

103

104

105 DGo’ DG 1.) Citrate Synthase 6.) Succinate Dehydrogenase 2.) Aconitase
7.) Fumarase 3.) Isocitrate Dehydrogenase 8.) Malate Dehydrogenase 4.) α-Ketoglutarate Dehydrogenase 9.) Overall reaction 5.) Succinyl-CoA Synthetase

106 Krebs Cycle Energetics
Reaction Enzyme DG°' (kJ/mol) 1 Citrate synthase -32.2 2 Aconitase +6.3 3 Isocitrate dehydrogenase -20.9 4 a-Ketoglutarate dehydrogenase complex -33.5 5 Succinyl-CoA synthetase -2.9 6 Succinate dehydrogenase 0.0 7 Fumerase -3.8 8 Malate dehydrogenase +29.7

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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