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

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

Phosphate Compounds Phosphate compounds in living organisms  High-energy compounds :  G’ o : <-25 kJ/mol  ATP:  G’ o = kJ/mol  Low-energy compounds :  G’ o : >-25 kJ/mol  Glucose-6-phosphate:  G’ o = 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 = kJ/mol (2) ADP + Pi  ATP + H 2 O ;  G’ o = kJ/mol Sum: PEP + ADP  pyruvate + ATP ;  G’ o = kJ/mol

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

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

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

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

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

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

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

13.3 Biological Oxidation-Reduction Reactions

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

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

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

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, 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 V/n ln [e - acceptor]/[e - donor] at 298K Standard reduction potential at pH 7.0, E’ o

Standard Reduction Potentials

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 = V  (2) NAD + + 2H + + 2e -  NADH + H +, E’ o = V  (1) - (2) = Acetaldehyde + NADH + H +  ethanol + NAD +   E’ o = E’ o of e - acceptor - E’ o of e - donor = V - ( V) = V   G’ o = -2 (96.5 kJ/V mol)(0.123V) = kJ/mol  1 M acetaldehyde and NADH, 0.1M ethanol and NAD +  E acetaldehyde = E’ o + RT/nF ln [acetaldehyde]/[ethanol] = V V/2 ln 1/0.1 = V  E NADH = E’ o + RT/nF ln [NAD + ]/[NADH] = V V/2 ln 1/0.1 = V   E = V - ( V) = V   G = -2 (96.5 kJ/V mol)(0.183V) = kJ/mol

e - carriers Complete oxidation of glucose  C 6 H 12 O 6 + 6O 2  6 CO 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

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

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

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

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 = 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

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

Enzymes with Electron Carriers