Term 2 Lecture 11: ATP And ATPase Flashcards
Metabolic reactions determine how exergonic and endergonic processes are coupled together
The stoichiometry of chemical reactions in metabolism determine how much ATP can be produced and how much ATP is required to synthesise biological molecules or carry out other processes
ATP must balance out within the cell, growth is possible by importing material/energy from outside
ATP is converted to ADP for
Movement, active transport,biosynthesis and signal amplification.
ADP is then converted back to ADP xia oxidation of fuel molecules or photosynthesis in aerobic organisms
Aerobic organisms need a lot of ATP to continue living infact we make and use nearly our whole body weight in ATP every day
ATP stability and importance
ATP is not stable thermodynamically because the ∆G for hydrolysis is negative under biological conditions. Therefore ATP hydrolysis occurs spontaneously.
BUT ATP is stable kinetically so ATP hydrolysis at pH 7.0 is slow due to high activation energy. The molecule has a half life of days under biological conditions.
^ this timescale is much longer than biological processes
ATP is sometimes referred to as a “high energy phosphate compound” but this is incorrect. It functions as an energy store by virtue of metabolism, not due to any special properties.
Some textbooks also draw ATP with a high energy bond between the second and third phosphate group this is also incorrect it is just a normal covalent bond.
ATP structure
ATP:
Adenosine (Adenine+ribose by glycosidic bond)
Bonded to a triphosphate residue by a phosphoric acid bond
Bonds between the 3 phosphate residues (alpha, beta and gamma) are phosphoric acid anhydride bonds
ATP hydrolysis and energy
ATP+H2O →ADP+Pi
∆G⁰’ = -30kJmol-¹
If ATP is hydrolysed without being “coupled” to another process the energy is lost as heat (useless to biological systems)
> So ATP hydrolysis is always linked to another process so that the chemical energy can be extracted in a usable form
E.g. Glucose+Pi →Glucose 6 phosphate is endergonic with a POS ∆G and is coupled to ATP→ADP+Pi with a large neg ∆G to give:
glucose+ATP→glucose 6 phosphate+ADP with an overall neg ∆G
E.g. ATP is used to phosphorylate an ion transport protein channel and drive the pumping of Na+ across a membrane.
-ATP is not hydrolysed directly
- instead ATP is used to phosphorylate the pump protein to drive confirmational changes (change in shape) resulting in an open and functioning ion channel
- energy is required as Na+ is pumped against the gradient
Alternative forms of stored energy
A balance of oxidising and reducing reactions must be maintained within cells
^ AKA redox conditions, if not maintained then metabolic reactions become impossible
Energy generation links redox reactions to ATP production - oxidation of reduced carbon compounds generates reduced NADH used to produce ATP
NADH (&NADPH) function as the cellular redox potential energy store (as do FADH2 and various other thiols e.g. glutathione to a lesser extent)
Reduced cofactors are a form of cellular energy as they are used to produce ATP - living cells have further energy stores that act as “rechargeable batteries”
↓
Chemical free energy potential in the form of other energy storage molecules e.g. GTP, creatine phosphate and PEP formed using ATP to drive synthetic reactions.
Stored nutrients e.g. starch and oils also act as chemical free energy potential and are synthesised in situ.
Establishment and maintenance of concentration gradients (usually across membranes). Can be used to generate ATP.
Establishment and maintenance of electrical potential from ion transport across membranes can also generate ATP in combination with concentration gradients.
Cells only maintain minimum reserves of ATP
E.g. muscle cells have only enough ATP stored to last for a second or two under load. ATP must be replenished by using stored creatine phosphate to produce ATP and then by metabolism to generate ATP.
Creatine phosphate can then be resynthesised from ATP.
ATP is continuously formed and broken down in active cells
ATP+creatine <-> ADP + creatine phosphate
(See graph start of notebook 3)
The F1F0 ATP Synthase
Enzyme that catalyses the formation of ATP from ADP. Present in aerobic prokaryotes and eukaryotic organelles derived from them.
The F0 part is always in a membrane facing into a compartment at a lower pH (higher H+ pos p phase)
The F1 part is always in a compartment corresponding to the bacterial cytosol (neg N phase)
P phase (F0 here)/ N phase (F1 here)
in bacteria
Periplasm/cytoplasm
In mitochondria
Intermembrane space/matrix
In chloroplasts
Thylakoid lumen/ outside membrane of thylakoid (stroma side)
Proton gradient
Bacterium:
Proton gradient low H+ inside ( in cytoplasm)
F0F1 ATPase takes in protons from outside and puts them inside
converting ATP →ADP
Process is coupled to use of reduced cofactors e.g. NADH converted to NAD+ as part of the electron transport chain.
^ which then pumps protons out again
Shape of ATP synthase
Has 2 multiprotein complexes F0 and F1
F0 is a transmembrane complex it contains multiple amphipathic helical subunits (10-14 C units make up the c ring) and a transmembrane subunit bound (non cov) to the outside of the ring containing a proton channel and two “stalk” subunits projecting into the matrix.
F1 is a water soluble globular complex in the matrix containing 3 pairs of subunits (alpha 3 beta 3) similar to NTPases linked to a central stalk with a 3 sided “axle” subunit (gamma lc). The attachment subunits (epsilon and delta lc) assist to hold the 2 subunits together.
Parts of the F0F1 complex can rotate
Both the rotor and stator parts of the F0F1 complex contain subunits derived from both F0 & F1 units.
Proton channel a, stalk subunits b2, delta subunit, and alpha 3 beta 3 are static
The c-ring, gamma “axle” and epsilon unit rotate. The rotation is clockwise (looking in from outside the cell) and the rotation of gamma causes it to bump into the alpha 3 beta 3 units distorting them, forcing beta subunits to take in an ADP and phosphate group (Pi) then release ATP forcing catalysis.
On the outside (P-phase) there are lots of protons. A proton (H+) enters the outer half channel of a subunit (proton channel) and is transferred to one of the c subunits where it is bound by an aspartate side chain. The proton travels all the way around the c ring and exits through a inner half channel (aka exit channel) into the cytosol (N- phase)
How do we know it rotates?
We can see it using a method developed by scientists in Japan in the 1990’s. The F1 part is broken off from the membrane part so that just the 3 alpha/beta subunits and the gamma stalk remain. The beta subunits are attached to a microscope slide by tags and a fluorescent tagged actin filament is attached to the top of the stalk. As membrane and proton gradients are gone this F1 part converts ATP to ADP causing the alpha/beta subunits to rotate the gamma stalk and the filament rotates with it in discrete 120⁰ steps visible via a fluorescent microscope
So what makes the c-ring rotate clockwise?
Driven by protonation of aspartate side chains in the c subunits.
Ionisation of the carboxylic acid group in the aspartate side chain Asp61 in subunit c determines hydrophobicity/ hydrophilicity:
RH2COO- + H+ <-> RH2COOH
RH2COO- is the charged form, it cannot move into the membrane and must face the proton channel in the a subunit.
RH2COOH is the protonated uncharged form that is relatively hydrophobic and can move into the membrane (amongst the fatty tails).
Addition of a proton from the intermembrane space can occur via the outer half channel of the a subunit of F0.
This allows the c subunit of F0 facing the channel to move into the membrane causing the rotor part of the complex to rotate clockwise eventually leading to a proton being passed from a c subunit into the inner half channel of the a subunit and from there into the N-phase region.
Rarely the c-ring rotates anticlockwise one step due to unfavourable conditions.
How rotation and ATP synthesis are linked
Beta subunits of the alpha 3 beta 3 hexameric assembly of F1 carry out catalysis.
Rotation of the asymmetric gamma subunit relative to the 3 beta subunits changes their confirmation (shape) and this leads to ATP synthesis
All 3 subunits cycle through the 3 states as the gamma protein stalk rotates forcing ATP synthesis.
The 3 states are:
O: open confirmation which can bind ADP+Pi or release ATP
L: loose confirmation that contains bound ADP+Pi
T: tight confirmation that catalyses ADP+Pi→ATP + H2O
Rotation clockwise synthesises ATP, rotation anticlockwise hydrolyses ATP
(See diagram start of notebook 3)
ATP synthase works on the basis of molecular motion
It is able to exploit random motion at the molecular level by means of a molecular “ratchet” (Aka a Brownian, Feynman or thermal ratchet)
Thermal as it utilises heat to drive motion
The ratchet and pawl prevent the c subunit ring of the F0 from turning in a random direction and constrain it to turn in one direction (most of the time.)
The ratchet and pawl is based on electrostatic interactions between charged species ATP synthase requires energy input from the flow of protons down the concentration and potential gradient to drive the process.
This “molecular motor” is a true nano-machine. It exploits processes at the molecular level. A direct “scale up” to normal size would not work but a “stepper” electrical motor uses a similar idea
