ATP Hydrolysis
Presented by: Rami Amro
Instructor: Dr. Neiman
• Ch. 13: Direct movement of the motors along the
filament thought to be a result of conformational
changes while it is attached.
• Ch.14:
• How changes in nucleotide state modify the
association of a motor with the filament.
• How these changes responsible for the working
stroke.
ATP:
• Was discovered in muscle extracts
(Lohman,1929)
• Was shown to be hydrolyzed by
actomyosine (Engelhardt &
ljubimowa,1939)
• It is the energy currency (Lipmann, 1941).
Hydrolysis of gamma phosphate
 ADP + Pi
ATP 
K eq 
[ADP]eq [ Pi ]eq
[ATP]eq
 4.9 105 M " at constant pH"
Where;
[ATP]  [MgATP 2- ]  [ MgATP - ]  [ MgATP]  [ATP 4- ]  ...
and, [ADP] &[Pi ] also refers to the sum of the concentration of the
species they exist in.
The equilibrium constant depends on:
1- free Magnesium concentration.
2-pH
3-ionic strength (mM).
Ex: [ ATP]  pM , [ADP]=[Pi ]~1mM, T=25  C
 K eq  4.9 105 M "Alberty & Goldberg, 1992"
* equilibrating process is extremely slow ~a week at 0  C , and so:
Gamma-phosphate bond is a high-energy intermediate state
Free energy of the Hydrolysis
reaction
Hydrolysis: is the release of energy from the High
energy ATP to be used for work, and producing low
level energy bonds.
G  G  kT ln
[ATP]c
, G  54 1021 J
[ADP]c [Pi ]
*at equilibrium G  0.
*G is the standard free energy.
* in cells :
[ATP]~ 1mM, [ADP]~10 M, [Pi ]~ 1mM  G  1011021 J  G
The hydrolysis reaction:
Transition state is a limiting speed step
Coupling chemical changes to
conformational changes.
• “How do nucleotides regulate the association of
the motor with the filament?” (Howard P:232)
• Motors Have four chemical states:
 M.T 
 M.D.P 
 M.D
M 
M  Neucleotide-free
M.T  ATP-bound
M.D.P  products-bound
M.D  ADP-bound
• Motors also have two mechanical states: attached and
detached to the filament.
•
so the number of states is 2*4=8.
• In fact they might be higher, since these states might
accompany more than one structural state. (Pre-stroke,
post-stroke).
• This implies there are multiple paths to the hydrolysis
cycle.
• The hydrolysis cycle defined by the most likely path.
• For two-headed motors the situation is more complicated
(64 states), but still there are special cases.
Hydrolysis of ATP by skeletal
Muscles Myosin.
• The release of phosphte is catalyzed by
the binding of myosin to actin.
• The release of myosin from actin is
catalyzed by the binding of ATP.
• Hydrolysis cycle happens :
1-with actin
2-and without actin.
Without Actin.
[ATP]=4 mM,[Pi]=2 mM, [ADP]=20µM “Muscle cell”
The binding of myosin and ATP or ADP is measured using stopped-flow
apparatus: change in the fluorescence.
ATP hydrolysis is measured using quenched-flow apparatus: mixed, then
quenched with acid to stop the hydrolysis reaction, then the amount of
nucleotide in its ATP, and ADP is measured.
• The hydrolysis reaction is reversible: 10% of the
nucleotide found in the ATP form (second step in
the cycle).
This happens as shown below:
1-ATP hydrolyze and release the gamma phosphate.
2-Gamma phosphate rotate while it is still attached
to the protein.
3- phosphate rebind to ADP, and make ATP.
• The rate at which free ATP produced is very small.
• Myosin has high affinity to ATP
• www.youtube.com/watch?v=a39WXFPB8E&feature=related
Hydrolysis with Actin
• Myosine binds strongly to Actin and
gamma-phosphate but not both at the
same time.
• Actin accelerate myosin’s ATPase 200folds
• So actin binding catalyze the release of
the phosphate “it was the limiting step”
How mechanical and chemical
cycles are coupled
• Binding to the actin filament catalyzes the
release of phosphate, then ATP binds to
catalyze the dissociation from the filament.
[actin]=1mM “Skeletal muscle”.
103S-1
20000S-1
≥104S-1
30S-1
100S-1
2000S-1
Explanation:
• Weakly bound state( low affinity for actin in the
ATP and ADP.Pi states ).
• Strongly bounded state (high affinity for actin in
the ADP and the nucleotide-free states).
• Weekly bound state (detached) has short life time
~10µs, so it doesn’t slow down the contraction.
• The release of phosphate after binding put the
motor in highly strained state A.M.D*, its
relaxation is the driving force for the filament to
slide.
Notes on the cycle with Actin
• myosin undergoes conformational changes before the
release of ADP.
Stroke happens earlier.
• Strained A.M.D* has 12kBT higher than the unstrained
A.M.D.” load slows the transition”
• In isometrically contracting muscle, A.M.D* is more
stable, and phosphate can easily binds.
• Adding more phosphate to unloaded muscle has no
effect on the contraction speed. D.const for Pi .~500M
• Binding of M.D.P to actin creates large force enough to
put the motor in the strained A.M.D* state.
• The weakly bound state may not exist in the mechanism.
Conclusions (Part1)
• Nucleotide chemistry:
1. Regulate the attachment and
detachment of the motors.
2. Drives the working stroke while the head
attached and detached.
3. The mechanochemical coupling is tight
“energy efficient”.