1. Product stabilizations in hydrolysis Relief of electrostatic repulsion by charge separation Ionization Isomerization Resonance

2. ATP Provides Energy by Group Transfers, not by Simple Hydrolysis Energy from group transfer  Two step reaction  Phosphoryl group transfer  Phosphoryl group displacement Energy directly from ATP hydrolysis  Hydrolysis of bound ATP (GTP)  protein conformational change  mechanical motion, activity transition (active  inactive)  Muscle contraction, movement of enzymes along DNA, movement of ribosome along mRNA, helicase, GTP-binding proteins

3. Phosphate Compounds Phosphate compounds in living organisms  High-energy compounds :  G’ o : <-25 kJ/mol  ATP:  G’ o = -30.5 kJ/mol  Low-energy compounds :  G’ o : >-25 kJ/mol  Glucose-6-phosphate:  G’ o = -13.8 kJ/mol Flow of phosphoryl group  From a compound with high phosphoryl group transfer potential to low potential (1) PEP + H 2 O  pyruvate + Pi ;  G’ o = -61.9 kJ/mol (2) ADP + Pi  ATP + H 2 O ;  G’ o = +30.5 kJ/mol Sum: PEP + ADP  pyruvate + ATP ;  G’ o = -31.4 kJ/mol

4. Nucleophilic Displacement Reactions of ATP Reactions of ATP  S N 2 nucleophilic displacements  Nucleophiles; O of alcohol or carboxylate/ N of creatine, Arg, His Nucleophilic attacks of the three phosphates   -  phosphoanhydride bond has a higher energy (~46 kJ/mol) than  -  (~31 kJ/mol)  PP i  2 P i by inorganic phosphatase  G’ o = -19 kJ/mol further energy “push” for the adenylylation reaction

5. Nucleophilic Displacement Reactions of ATP Energy-coupling mechanism via adenylylation reaction

6. Bioluminescence of Firefly Conversion of chemical energy to light energy using ATP

7. Assembly of Informational Macromolecules Requires Energy DNA or RNA synthesis  NTP  release of PPi and hydrolysis to 2 Pi Protein synthesis  Activation of amino acid by adenylylation

8. ATP Energizes Active Transport and Muscle Contraction ATP for molecular transport  2/3 of the energy at rest is used for Na + /K + pump in human kidney and brain Contraction of skeletal muscle  ATP hydrolysis in myosin head  Movement along the actin filament

9. Transphosphorylation Between Nucleotides NTPs and dNTPs  Energetically equivalent to ATP  Generation from ATP Nucleoside diphosphate kinase  ATP + NDP (or dNDP) ADP + NTP (or dNTP)   G’ o ≈ 0  Driven by high [ATP]/[ADP]  Ping-pong mechanism

10. Transphosphorylation Between Nucleotides Adenylate kinase  2ADP ATP + AMP,  G’ o ≈ 0  Under high ATP demanding conditions Creatin kinase  ADP + PCr ATP + Cr,  G’ o = -12.5kJ/mol  PCr : Phosphoryl reservoir for high ATP demanding conditions in muscle, brain, and kidney Inorganic Polyphosphate (PolyP) as a Phosphate Group Donor  PolyP  Polymer of phosphate  Phosphagen: reservoir of phosphoryl groups  In prokaryotes  PolyP kinase-1 : synthesis of polyP  ATP + polyP n ADP + polyP n+1  PolyP kinase-2 : synthesis of GTP or ATP  GDP + polyP n+1 GTP + polyP n

11. 13.3 Biological Oxidation-Reduction Reactions

12. Electron flow can do biological work Electromotive force (emf)  Force proportional to the difference in electron affinity between two species  “Do work”  Glucose (e - source)  sequential enzymatic oxidation  e - release Flow e - via e - carriers  O 2  e.g. generation of proton motive force in mitochondria to generate ATP emf; provide energy for biological works

13. Oxidation States of C in the Biosphere Oxidation state of C  Number of electrons owned by C  depends on electronegativity of bonding atoms  O> N> S> C> H  In biological system; biological o xidation = dehydrogenation

14. Biological Oxidation Often Involve Dehydrogenation 4 ways for electron transfer  Direct electron transfer via redox pairs Fe 2+ + Cu 2+  Fe 3+ + Cu +  Transfer of hydrogen atoms AH 2  A + 2e - + 2H + AH 2 + B  A + BH 2  Transfer of hydride ion (:H - )  Direct combination with O 2 R-CH 3 + 1/2 O 2  R-CH 2 -OH Reducing equivalent  A single e - equivalent participating in an oxidation-reduction reaction  Biological oxidation  Transfer of two reducing equivalents

15. Reduction Potential measures for e - affinity Standard reduction potential, E o  A measure of affinity of electron  Measurement of E o  Standard reference half reaction (hydrogen electrode) H + + e -  1/2H 2, E o = 0 V  Connection of the hydrogen electrode to another half cell (1M of oxidant and reductant, 101.3 kPa)  The half cell with the stronger tendency to acquire electrons : positive E o Reduction potential E  E = E o + RT/nF ln [e - acceptor]/[e - donor]  n: the number of e - transferred/molecule  F; Faraday constant  E = E o + 0.026V/n ln [e - acceptor]/[e - donor] at 298K Standard reduction potential at pH 7.0, E’ o

16. Standard Reduction Potentials

17. Free Energy Change For Oxidation Reduction Reaction Oxidation-reduction reaction  The direction of e - flow ; to the half-cell with more positive E   G = -nF  E or  G’ o = -nF  E’ o Calculation of  G  Standard conditions, pH 7, 1M of each components  (1) Acetaldehyde + 2H + + 2e -  ethanol, E’ o = -0.197 V  (2) NAD + + 2H + + 2e -  NADH + H +, E’ o = -0.320 V  (1) - (2) = Acetaldehyde + NADH + H +  ethanol + NAD +   E’ o = E’ o of e - acceptor - E’ o of e - donor = -0.197 V - (-0.320 V) = 0.123 V   G’ o = -2 (96.5 kJ/V mol)(0.123V) = -23.7 kJ/mol  1 M acetaldehyde and NADH, 0.1M ethanol and NAD +  E acetaldehyde = E’ o + RT/nF ln [acetaldehyde]/[ethanol] = -0.197 V + 0.026 V/2 ln 1/0.1 = -0.167 V  E NADH = E’ o + RT/nF ln [NAD + ]/[NADH] = -0.320 V + 0.026 V/2 ln 1/0.1 = -0.350 V   E = -0.167 V - (-0.350 V) = 0.183 V   G = -2 (96.5 kJ/V mol)(0.183V) = -35.3 kJ/mol

18. e - carriers Complete oxidation of glucose  C 6 H 12 O 6 + 6O 2  6 CO 2 + 6 H 2 O   G’ o = -2,840 kJ/mol  e - removed in oxidation steps are transferred to e - carriers Electron carriers  NAD +, NADP + : soluble carrier  FMN, FAD : prosthetic group of flavoproteins  Quinones (ubiquinone, plastoquinone) : membrane  Iron-sulfur proteins, cytochromes : cytosol or membrane

19. NADH and NADPH NAD(P)  Nicotinamide adenine dinucleotide (phosphate)  Accept hydride (:H - ) released from oxidation (dehydrogenation) of substrate : either A side or B side, not both sides  NAD(P) + + 2H + + 2e -  NADH + H +  NAD(P) + : + indicates oxidized form, not the net charge of NAD(P) which is - Benzenoid ring Quinonoid ring

20. Metabolic Roles of NADH and NADPH  NADH  Functions in oxidations in catabolic reactions (NAD + > NADH)  NADPH  Functions in reductions in anabolic reactions (NADPH > NADP + ) Oxidoreductases or dehydrogenases  Specific preference to NAD or NADP  Spatial segregation  Oxidation of fuels in mitochondria  Biosynthesis in cytosol  AH 2 + NAD +  A + NADH + H +  Alcohol dehydrogenase  CH 3 CH 2 OH + NAD +  CH 3 CHO + NADH + H +  A + NADPH + H +  AH 2 + NADP + Rossmann fold  NAD or NADP binding domain  Relatively loose binding  diffusion to other enzyme

21. Dietary Deficiency of Niacin: Pellagra Niacin (nicotinic acid)  Source of the pyridine-like ring of NAD and NADP  Low amount of synthesis from Trp in human  Pellagra (rough skin)  Disease from niacin deficiency  Can be cured by niacin or nicotinamide, not by nicotine

22. Flavin Nucleotide Flavin nucleotides  FMN : flavin mononucleotide  FAD: falvin adenine dinucleotide  Derived from riboflavin  One or two electron transfer Flavoproteins  Enzymes catalyzing oxidation-reduction reactions using FMN or FAD as coenzyme  Tight-bound flavin nucleotides  Different E’ o from that of free flavin nucleotide  FAD in succinate dehydrogenase : E’ o = 0 V  Free FAD : E’ o = -0.219 V  Some contains additional tightly bound inorganic ions (Fe or Mo)  c.f. Cryptochromes  Flavoproteins mediating the blue light effect in plant and controlling circadian rhythms in mammals

23. Flavin Nucleotide 360nm absorption 450nm absorption 370 and 440 nm absorption

24. Enzymes with Electron Carriers