ATP Hydrolysis. Presented by: Rami Amro Instructor: Dr. Neiman

Transcription

1 ATP Hydrolysis Presented by: Rami Amro Instructor: Dr. Neiman

2 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.

3 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).

4 Hydrolysis of gamma phosphate ATP ADP + Pi [ADP] eq[ P i] eq 5 Keq M " at constant ph" [ATP] Where; eq [ATP] [MgATP ] [ ATP ] [ ATP] [ATP ]... Mg Mg and, [ADP]&[P ] also refers to the sum of the concentration of the i species they exist in. The equilibrium constant depends on: 1- free Magnesium concentration. 2-pH 3-ionic strength (mm). Ex: [ ATP] pm, [ADP]=[P ]~1mM, T=25 C K eq i "Alberty & Goldberg, 1992" M * equilibrating process is extremely slow ~a week at 0 C, and so: Gamma-phosphate bond is a high-energy intermediate state

5 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. [ATP] ln, c 21 G G kt G J [ADP] c[p i] *at equilibrium G 0. * G is the standard free energy. * in cells : [ATP]~ 1mM, [ADP]~10 M, [P ]~ 1mM i 21 G J G

6 The hydrolysis reaction: Transition state is a limiting speed step

7 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 M.T M.D.P M.D M Neucleotide-free M.T ATP-bound M.D.P products-bound M.D ADP-bound

8 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.

9 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.

10 Without Actin. [ATP]=4 mm,[p i ]=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.

11 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 XFPB8E&feature=related

12 Hydrolysis with Actin Myosine binds strongly to Actin and gamma-phosphate but not both at the same time. Actin accelerate myosin s ATPase 200- folds So actin binding catalyze the release of the phosphate it was the limiting step

13 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.

14 103S S S -1 30S S S -1

15 Explanation: Weakly bound state( low affinity for actin in the ATP and ADP.P i 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.

16 Notes on the cycle with Actin myosin undergoes conformational changes before the release of ADP. Stroke happens earlier. Strained A.M.D* has 12k B T 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 P i. ~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.

17 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.