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 = -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 + H2O pyruvate + Pi ; G’o = -61.9 kJ/mol(2) ADP + Pi ATP + H2O ; G’o = +30.5 kJ/molSum: PEP + ADP pyruvate + ATP ; G’o = -31.4 kJ/mol

Nucleophilic Displacement Reactions of ATP

Reactions of ATP SN2 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)

PPi 2 Pi 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 + polyPn ADP + polyPn+1

PolyP kinase-2 : synthesis of GTP or ATP GDP + polyPn+1 GTP + polyPn

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 O2

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 oxidation = dehydrogenation

Biological Oxidation Often Involve Dehydrogenation

4 ways for electron transfer Direct electron transfer via redox pairs

Fe2+ + Cu2+ Fe3+ + Cu+

Transfer of hydrogen atomsAH2 A + 2e- + 2H+

AH2 + B A + BH2

Transfer of hydride ion (:H-) Direct combination with O2

R-CH3 + 1/2O2 R-CH2-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, Eo

A measure of affinity of electron Measurement of Eo

Standard reference half reaction (hydrogen electrode)

H+ + e- 1/2H2 , Eo = 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 Eo

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

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 = -nFE or G’o = -nFE’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+

Eacetaldehyde = E’o + RT/nF ln [acetaldehyde]/[ethanol] = -0.197 V + 0.026 V/2 ln 1/0.1 = -0.167 V ENADH = 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

e- carriers

Complete oxidation of glucose C6H12O6 + 6O2 6 CO2 + 6 H2O 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

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

AH2 + NAD+ A + NADH + H+

Alcohol dehydrogenase CH3CH2OH + NAD+ CH3CHO + NADH + H+

A + NADPH + H+ AH2 + 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 = -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

Flavin Nucleotide

360nm absorption

450nm absorption

370 and 440 nm absorption

Enzymes with Electron Carriers