16 “Phosphate Transfer Potential” is a fancy-schmancy term for –DGo’
17 Electrochemistry in review One beaker w/ ZnSO4 and a Zn electrodeOne beaker w/ CuSO4 and a Cu electrodeZinc gets oxidized and the electrode slowly vanishesCopper gets reduced and the electrode gets fatter
19 Redox Table Higher the SRP, the better the oxidant Lower the SRP, the better the reductantAny substance can oxidize any substance below it in the table.The number of reactants involved doesn’t change the reduction potentiali.e. if a reaction involves 2 NAD+, the SRP is still V
20 Electrochemistry in review Zinc gets oxidizedCopper gets reducedWhat determines who gets oxidized?
21 DEo and Keq For an actual half reaction aA + ne- ⇌ aA- For an actual redox reaction:A+n + ne- ⇌ AB ⇌ B+n + ne-A+n + B ⇌ A + B+nand(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 DGoSo:But we already know:Therefore:Another Point!
24 NAD+ Reduction (Nicotinamide Adenine Dinucleotide) NAD+ is a common redox cofactor in biochemistry
25 Coenzyme QCoenzyme Q is another electron carrier in the cell
26 An Example:What is DGo’ for theOxidation of NADH by Ubiquinone?
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.
40 Don’t Eat the Toothpaste! Phosphoglucomutase contains a PO4-2 group attached to residue D8.Fluoride has a number of toxic effectsOne of them is the removal of the phosphate from phosphoglucomutaseNo phosphate = no activityNo activity = can’t utilize glycogen
46 Aldolase ReactionThe 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
49 Phosphoglyceromutase H8 in human erythrocyte PGM
50 Overall ReactionThe 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 2003Mono- and disodium methyl arsenate are used as agricultural insecticidesArsphenamine was one of the first treatments for syphilisArsenic trioxide is an approved treatment for promyelocytic leukemiaLewisite is an old-fashioned CBW blister and lung agentCoppers acetoarsenite is “Paris green,” a pigment used by artists, some of whom had the habit of licking their brushesScheele’s Green (copper arsenite) was used as a coloring agent for candy in the 19th century
56 Antitrypanosomals Chagas Disease African Sleeping Sickness Nagana Leishmaniasis (“Baghdad Boil”)Afflict hundreds of millionsNagana responsible for the popularity of cannibalism in the African “fly belt.”Leishmaniasis is now endemic in TexasRemember 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 enzymesGood drug targets
58 AntitrypanosomalsModel of L. mexicana glyceraldehyde-3-phosphate dehydrogenase complexed with N6-(1-naphthylmethyl)-2¢-deoxy-2¢- (3-methoxybenzamido)-adenosine.
59 AntitrypanosomalsBinding 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 zero3 of 10 Rxns have large, negative DG (i.e. irreversible)Large negative DG Rxns are sites of regulation!
62 Hexokinase regulation Hexokinase – muscleKm for glucose is 0.1 mM; cell has 4 mm glucoseSo hexokinase is normally active!Allosterically inhibited by (product) glucose-6-P (product inhibition)Glucokinase – liver, pancreasKm glucose ≈ 8 mM (144 mg/dl – above normal)Cooperative – nH ≈ 1.7No product inhibitionOnly turns on when cell is rich in glucoseShifts hepatocytes from “fasting” to “fed” metabolic states, encouraging glycogen synthesis and glycolysisActs as signal in pancreas to release insulin
64 PFKPFK 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 F6PAMP, 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
69 Glucose Catabolism Part 2 Pyruvate Dehydrogenase Huge multienzyme complex4.6 Mdaltons in E. Coli (a24b24g12)9 Mdaltons in mammals (a60b60g24)3 separate enzyme functions create overall reactionPyruvate + NAD+ + HSCoA CO2 + Acetyl CoA + NADHThis is where we actually lose our first carbon(s) from glucose
74 E1 – Pyruvate Dehydrogenase Proper In E. coli, E1 is a dimer of two similar subunitsIn mammals, E1 is an a2b2 tetramer.Each E1 contains 2 active sitesEach 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
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 - RegulationRegulation 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.
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.
93 At EquilibriumCitrate91%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 chiralMultipoint binding allows stereospecificity in a nonchiral compound
95 An Aconitase Inhibitor Sodium Fluoroacetate is a fairly potent toxin (2-10 mg/kg)Brand name 1080Incoporated into fluoroacetylCoA, then into fluorocitrateFluorocitrate is a powerful competitive inhibitor of aconitase
97 Isocitrate Dehydrogenase DGo’ = kJ/molOxidation: NAD+ oxidizes the hydroxyl carbon of isocitrateDecarboxylation: A Mn+2 bound to the enzyme stabilizes the intermediateProtonation: Reforms the carbonyl to generate productGeneral 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 directlyCritters make GTP, then exchange phosphate to ATP
100 Succinyl CoA Synthetase Rxn CoA is displaced by an OrthophosphateThe phosphate group is transferred to a Histidine residue on the enzymeSuccinate leaves as a productThe 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.