lecture 25 the sources of energy: posphorylation, oxidation and coupling chemical energy to work
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Lecture 25
The sources of energy: posphorylation, oxidation and coupling chemical energy to work
Peter Mitchell
Why protons?
Why ATP?
Why oxygen?
Most cells use a proton gradient as an energy source across their plasma membrane. Why do animal cells use a sodium gradient?
Why protons?
Protons because one single Histidine, Glutamate or Aspartate residue furnish a simple and tunable binding site for H+. pKa of these groups can vary by 2-3 units depending on the environment. These sites do not bind metal ions tightly unless work in concert.
It is much more difficult to build a selective binding site that would discriminate between Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Pb2+, Hg2+, Fe2+, Fe3+, etc
Protomotive Force has an electrical component and can couple electrochemical proton gradient to the transport of other charged substances
mVinpHFH .....60
JoulesinFH
HRTH ...)
][
][ln(
2
1
-180 mV
pH = 8
0 mV
pH = 7
PMF = -60 -180 = -240 mV
Measuring the membrane potential…..
-180 mV
pH = 8
0 mV
pH = 7
Fluorescent dye Rhodamine 123 (Rh123+) will penetrate into the vesicles according to electric gradient, increasing their fluorescence. Increased concentration of Rh123 inside the vesicle beyond certain point will cause self-quenching.
positive charge
Calibration???Rhodamine 123
Measuring the membrane potential…..another way
-180 mV
pH = 8
0 mV
pH = 7
Valinomycin…potassium uniporter
K+
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm
What happens if we add valinomycin?
)][
][ln(
in
out
K
K
F
RT
but val. may affect the potential…
Measuring the membrane potential…..a better way
time
fluor
esce
nce
val
0.1
0.05
0.2
[KCl] outside mM
Null method
Why ATP?
The reaction of ATP hydrolysis is very favorable
ΔGo = -30.5 kJ/mol = - 7.3 kCal/mol
because:
1. Charge separation of closely packed phosphate groups provides electrostatic relief
2. Inorganic Pi, the product of the reaction, is immediately resonance-stabilized (electron density spreads equally to all oxygens)
3. ADP immediately ionizes giving H+ into a low [H+] environment (pH~7)
4. Both Pi and ADP are more favorably solvated by water than one ATP molecule.
ATP exists in complex with Mg2+
Mg2+
phosphoanhydride bonds
ATP is not the only:
Phosphoenolpyruvate (PEP) -61.9 kJ/mol
1,3-Bisphosphoglycerate -49.3 kJ/mol
Phosphocreatinine -43.0 kJ/mol
ATP -30.5 kJ/mol
Pyrophisphate (Pi-Pi) or
Inorganic polyphosphate (polyPi) -19 kJ/mol
Thioesters (Acetyl CoA) -31 kJ/mol
[ATP]
[ADP][P]lnRTGG o
p
Calculate Gp in erythrocytes if
Gp = -30.5 kJ/mol [ATP] = 2.3 mM;
[ADP] = 0.25; [Pi] = 1.65 mM,
In other cells: [ATP] [ADP] [AMP] [Pi] in mM
Rat myocyte 8 0.9 0.04 8.05Rat neuron 2.6 0.73 0.06 2.7E. coli 7.9 1.04 0.82 7.9
In real cells G for ATP hydrolysis is more negative than standard Go
ATP provides energy to group transfer reactions:
A-P → A + P G1
B-P → B + P G2
A-P + B → A + B-P G = G1-G2
ATP
1,3-BPGPEP
PEP
Phosphocreatinie
Glucose-6-P Glycerol-P
Pi
G o
f hy
drol
ysis
Synthesis of any phosphorylated compound can be coupled to ATP hydrolysis
Transfer reactions:Phoshoryl transfer; Pyrophosphoryl transfer; Adenylyl transfer
Synthesis of NTPs (dNTPs) from ATP occurs as phosphoryl exchange at G ~0. The reaction is catalyzed by nucleoside diphosphate kinase which first phosphorylates its own His, releases ADP and then phosporylates the incoming NDP or dNDP.
How is the energy of phosphoryl transfer (or removal) imparted to a conformational change or how the chemical work is done?
Simple binding of ATP to an enzyme (or another effector protein) may cause massive conformational change by allosteric mechanism through the stage of binding site rearrangement. Cleavage of the gamma phosphate would lead to another conformational change. A complete release of the nucleotide diphosphate returns the protein to its initial state.
Phosphorylation of certain sites (Tyr, Ser or Thr) promotes recognition by a counterpart domain (recall SH2 domains)
Why oxygen?
Electron re-distribution from less electronegative to more electronegative atoms occurs with massive energy release:
Electronegativity of common elements:
H < C < S < N < O …Cl < F
Identify the substance and the reduction state of the first carbon (red)
Enthalpies of oxidation (combustion)
hydrogen (MW 2)H2 + ½O2 → H2O -286 kJ/mol
methane (MW 16)CH4 + 3O2 → CO2 + 2H2O -891 kJ/mol
glucose (MW 180.2)C6H12O6 + 6O2 → 6CO2 + 6H2O -2840 kJ/mol
(-680 kCal/mol)palmitic acid (MW 256.4)C16H32O2 + 23O2 – 16CO2 + 16H2O - 9730 kJ/mol
Oxidation-reduction (Red-ox) reactions usually lead to re-distribution of electron densities or complete transfer of electrons resulting in change of ionization state.
Fe2+ + Cu2+ → Fe3+ + Cu+
or in the form of half-reactions: Fe2+ → Fe3+ + e
Cu2+ + e → Cu+
In biological systems oxidation is often coupled to dehydrogenation.
1. Direct transfer of electrons
2. As a transfer of H atoms or removal of H atoms coupled to production of H+
3. As a Hydride ion :H–
4. Through direct combination with oxygen
R-CH3 + (½)O2 → R-CH2-OH
Standard Reduction potentials for some half-reactions, Volt
½ O2 + 2H+ + 2e → H2O +0.816
Fe3+ + e → Fe2+ +0.771
Cytochrome c (Fe3+) + e → Cytochrome c (Fe2+) +0.254
Fumarate2- +2H+ + 2e → succinate2- +0.031
2H+ + 2e → H2 (standard condition) 0
Pyruvate + 2H+ + 2e → lactate -0.185
FAD + 2H+ + 2e → FADH2 -0.219
S + 2H+ + 2e → H2S -0.243
NAD+ + H+ + 2e → NADH -0.320
NADP+ + H+ + 2e → NADPH -0.324
α-ketoglutarate + CO2 + 2H+ + 2e → isocytrate -0.38
2H+ + 2e → H2 (pH 7) -0.414
Reduction potentials for mixtures of reductant/oxidant (hlf-reaction potentials) are measured using the standard hydrogen electrode
http://www.chemguide.co.uk/physical/redoxeqia/eomgdiag.gif
2H+ + 2e → H2 Mg2+ + 2e → Mg (metal)
donor][electron
acceptor][electron ln
nF
RTEE o
Electron transport chain puts reductants and oxidants in specific order
Complex I II III IV
Energy released in forming water is stored as a PMF
Energy is divided into smaller units ~12 protons per water molecule
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