ATP is the main energy source for almost all ATP synthase chemical processes in living systems
Protein synthesis DNA replication
ATP Muscle contraction DNA injection
Membrane bound F Soluble F1 ATPase 0
Why do we consume so much ATP? ATP, universal energy carrier of living systems Many reactions in biological cells are thermodynamically unfavorable (DG > 0). Hydrolysis A thermodynamically unfavorable reaction can be driven by a favorable one, if they are coupled.
A « B D G°' = +4 kcal/mol
(- D G°'/1.36) (- 4/1.36) -3 Synthesis K’ eq = [B]/[A] = 10 = 10 =1.15 ´ 10
No spontaneous formation of B, when [B]/[A] > 1.15 ´ 10-3, ATP + H 2O ADP + Pi so most of A remains unconverted. 0 DG = -12.2 kB T = -30.6 kJ/mol = -7.3 kcal/mol We can make much more of B if we couple A « B with a DG = DG 0 + ln ([ADP][P ]/[ATP]) = -13.7 kcal/mol favorable reaction. i ~1/500
ATP hydrolysis shifts the equilibria ATP synthase is the ATP factory which of coupled reactions synthesizes most of ATP in organisms
Coupled reaction: A « B D G°’ = + 4.0 kcal/mol Bacteria ATP synthase + ATP + H2O « ADP + Pi + H D G°’ = - 7.3 kcal/mol ------ATP synthase Mitochondria + A + ATP + H2O « B+ ADP + P i + H D G°’ = -3.3 kcal/mol
[B]eq [ADP] eq [Pi] eq ( - D G° ' / 1.36) (- (-3.3) / 1.36 ) 2 K'eq = ------• ------= 10 = 10 = 2.67 ´ 10 [A]eq [ATP] eq
[B]eq [ATP] eq ----- = K 'eq ------[A] = [ADP] [P ] eq eq i eq Chloroplast
If the cell keeps it’s [ATP] / ([ADP] [Pi ]) ratio at about 500, 2 5 [B] eq/ [A] eq = 2.67 ´ 10 ´ 500 = 1.34 ´ 10 , most of A has been converted!
Without ATP hydrolysis, this was 1.15 ´ 10-3, so A « B conversion has 8 been increased by a factor of 10 . ATP synthase
1 ATP synthase Structural Data ATP synthase is a rotary motor that couples proton translocation to ATP synthesis
Cytoplasm Yeast Stator
E. coli
~ 20 nm
H+ Periplasm Rotor
ATP synthase is a rotary motor that couples One shaft, two motors
proton translocation to ATP synthesis ~100Å ADP + Pi Cytoplasm F 1-ATPase Soluble part, F1-ATPase -Synthesizes ATP when torque is ATP applied to it (main function of this unit) ~80 Å - Produces torque when it hydrolyzes ATP - 5 subunits: 3a , 3 b , 1g, 1d and 1e ~200 Å
~ 20 nm Membrane-bound part,
F0 Complex ~60 Å - Produces torque when positive proton gradient across membrane F o complex (main function of this unit) - Pumps protons when torque is applied ~60 Å - 3 subunits: 1a, 2b and 9-12 c). Torque is transmitted between the motors via the central stalk. Periplasm H+ “ATP synthesis” requires both F0 and F1 parts.
Direct observation of ATP synthase rotary motion ATP synthesis mechanism
The applied torque causes rotation of the g -subunit which causes cyclic transformation of three catalytic sites.
aE
bE bDP
aDP
aTP
bTP
Bottom view We have six nucleotide binding sites, 3 catalytic and 3 non-catalytic
2 a and b subunits are similar in secondary structures Catalytic Nucleotide Binding Site b -Glu188 makes H-bonds to a water molecule and has it Water molecule is ready to attack the Mg 2+ b-barrel H-bonded by b-Glu188. g-phosphate of ATP domain
Mg 2+ Mg 2+ a/b domain
AMP-PNP AMP-PNP
a domain
aTP b TP nucleotide a -Arg 373 helps stabilize the 20% sequence identity. a TP-b TP interface negative charge - when isolated, hydrolyse ATP very slowly.
a -Gln208 corresponds Non-Catalytic Nucleotide Binding Site to Glu188 in b -subunit. It makes a -subunit A rough idea of central stalk’s tasks Water molecule non-catalytic Mg 2+ TP -> E -> DP -> TP Interpolation of observed states
b -Arg372 and b -Tyr368 nucleotide side chains which help b E-aTP interface forming the binding pocket move out of the pocket. aTP b TP
Assembling ATP Synthase F1 Torque application to F1
Torque is applied to the central stalk atoms at the F1-Fo interface to constrain their rotation to constant angular velocity w = 24 deg/ns.
central stalk, gde
applied torque
0.0 to 5.0 ns (0 to 120 deg) of torqued F1 rotation, w = 24 deg/ns.
3 Stalk analysis Different interactions between the central stalk and various b-subunits
Slowed torque transmission along central stalk
push this active site open b TP bE closes t = 3 ns b Eint DP (kcal/mol)
t = 0
average rotation (deg) b this active site less affected E bTP opens close this active site
time (ns) time(ns) stalk height (Å)
bDP does neither
Rotation Produces Synthesis-like Events (3) Motion of a Arg373 towards At 3.0 ns (72 deg) of rotation, we observe: TP • slowed torque transmission along central stalk bTP phospate binding pocket • unbinding from ATP at the b TP catalytic site
6.0 bTPThr163 -OG to 5.4 bTPPg
4.8 7000
4.2 ) Å 3.6 3500 bTPArg189 -Cz
distance( to (pN nm) bTPPg Applied torque
1 2 3 4 5 6 7 time (ns) time(ns)
QM/MM calculation of ATP hydrolysis Initial configuration
Transition state
Intermediate structure
4 Intermediate structure Schematic view of ATP hydrolysis in b TP
reactant state transition state
intermediate state product state
Product
Energetics of ATP hydrolysis in b TP Cross-linking has been used to determine positions of a- and b-subunit relative to
the c10-ring
The overall ATP hydrolysis reaction is found to be endothermic.
This suggests that bTP favors binding of ATP over product and hence corresponds to the tight conformation.
Presence of the protein environment leads to a substantial reduction in barrier height at the TS from 56 kcal/mol (gas phase) to 28 kcal/mol (protein).
The final product conformation is separated by a “rugged” energy barrier from the final product conformation and only slightly lower in energy.
Jones et. al, JBC , 275(2000)31340
a-subunit contains a proton path and aR210 is Ten c-subunits form c10 ring which is a rotor and essential for proton translocation D61 is essential for proton transfer
D61
aR210
D61
c1 c2
5 Two disconnected half water channels are formed during MD simulation
Cytoplasm Exit channel
4 nm
Periplasm Entrance Channel The proton pathway are formed by bound water and polar side chains of subunit a. It goes half way through subunit a and then another half way through the interface between subunit a and c-ring.
D61 is pulled out by R210 and its proton becomes exposed to one of the half channels
R210(+)
D61(-)
NMR structure of c-subunit at pH 8 shows that cTMH-2 Rotary Motions of Membrane unit is rotated by 140 o compared to that at pH 5 of ATP-synthase
cTMH -2 cTMH -1 Deprotonated D61
cTHM-1
Protonated D61 cTMH -2
pH 8 Steered Molecular Dynamics simulation of pH 5 single-helix rotation in the trans- membrane unit of ATP-synthase Girvin et. al, Biochemistry, 37(1998)8817 Rastogi et. al, Nature , 402(1999)263
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