ATP biosynthesis

Question 1

what evidence supports the existence of the chemiosmotic gradient

Answer 1

oxphos requires an intact IMM
IMM is impermeable to ions
electron transport creates a measurable electrochemical gradient across IMM
compounds that increase the permeability of the IMM allow electron transport but inhibit ATP synthesis

Question 2

what drives protons back into the matrix providing the energy for ATP synthesis

Answer 2

proton-motive force created by the electrochemical gradient

Question 3

what is the pH of the IMS and the matrix

Answer 3

IMS: 7
matrix: 8

Question 4

what conditions does mitochondrial ATP synthase play a key role in

Answer 4

cancer, obesity, neuropathies, Alzheimer’s and Parkinson’s, microbial infections, mitochondrial disease, immune defiency, cycstic fibrosis, diabetes and hypothyroidism

Question 5

which region of ATPase is intramembrane

Answer 5

C-ring domain

Question 6

what does the rotation of ATP synthase cause

Answer 6

the formation of ATP by bringing ADP and a phosphate closer together

Question 7

what types of energy are converted into one another in oxphos

Answer 7

electrochemical energy in the gradient
mechanical energy in the rotation of enzyme
chemical energy in the formation of ATP

Question 8

what method is primarily used to study ATP synthase function

Answer 8

structural method using cryo-EM to make a 3D model

Question 9

what is the F0 and F1 domain of ATP synthase
where are they

Answer 9

F0: intramembrane C domain structure forms a channel that allows flow of protons
F1: motor domain that protrudes into the matrix - rotation of central stalk causes conformational change of the 3 active sites

Question 10

what is the stator of ATP synthase
what is its role

Answer 10

structural support
prevention of unwanted rotation
transmembrane spanning
integration with the membrane

Question 11

where is the substrate channel of ATP synthase

Answer 11

ADP and Pi

Question 12

what is the importance of substrate channelling of ATP synthase

Answer 12

channelling substrates directly to the active sites:
optimized ATP synthesis, prevents wasteful hydrolysis of ATP, control of reaction pathway

Question 13

how many C subunits make up the C ring of the F0 region in human ATP synthase

Answer 13

8

Question 14

which regions of ATP synthase rotate

Answer 14

intramembrane C ring and central rod

Question 15

where is the DP site of ADP binding in ATP synthase

Answer 15

between subunit alpha (chain C) and beta (chain D)

Question 16

describe the F1 region of ATP synthase

Answer 16

3 alpha and 3 beta alternating domains around the central rotating gamma domain

Question 17

where is ATP synthase located in eukaryotes and in prokaryotes

Answer 17

EU: IMM
PRO: plasma membrane

Question 18

what is the pKa of glutamate residues

Answer 18

4.25

Question 19

how was the substrate binding sites of ATP synthase identified

Answer 19

structural studies

Question 20

where is the active site of ATP synthase

Answer 20

between the alpha and beta subunits (chain C and D)

Question 21

how was the function of ATP synthase determined using structural studies

Answer 21

a series of conformational changes can be identified from structural information which tell us how the enzyme functions
Rotary catalysis of ATP generation characterised by structural information

Question 22

why does the C-ring only rotate in one direction during ATP synthesis

Answer 22

unidirectional rotation is crucial for ATP synthesis
if it went the other way it would hydrolyse ATP
structure and mechanical constraints

Question 23

which residue acts as a proton transporter across the IMM

Answer 23

glutamate

Question 24

what causes deprotonation and deprotonation

Answer 24

Deprotonation occurs when the environment is at a higher pH than the pKa of the residue
protonation occurs when the environment is at a lower pH than the pKa of the residue

Question 25

what causes the C ring to rotate

Answer 25

protonated glutamate has a positively charged carboxyl group
this repels arginine residues in the stator region
causes it to rotate away

Question 26

what causes the transported proton to be released into the matrix from the C-ring

Answer 26

change in the local pKa causes deprotonation
proton repelled by arginine residues into the matrix

Question 27

explain the overall mechanism

Answer 27

as protons move through the F0 unit they cause it and the attached central stalk to rotate
rotation of the stalk causes conformational changes in the F1 unit
ADP and Pi are pushed together forming ATP

Question 28

explain oligomycin mechanism of action