Metabolism- Metabolic poisons Flashcards
Why was succinate added to the particles in the ATP synthesis assay?
To provide a substrate for the mitochondria to oxidise. This provides the proton motive force that powers ATP synthesis as shown below.
Why are the mother’ symptoms much less severe than her children’s?
This is a gene dosage effect. mtDNAis present within cells in multiple copies, some of which are mutated and some which are not (heteroplasmy).
The children of the mother have a greater percentage of mutated mtDNAthan their mother. In the original study this was analysedby a PCR reaction (shown below). The resulting band of 551bp is sensitive to digestion with the restriction enzyme AvaIifit contains the mutation. Densitometricanalysis of the bands in the gel can reveal the percentage of mutant DNA.
Why was succinate omitted from the second assay
To examine ATP hydrolysis in the absence of ATP production
What can you deduce about the nature of this mutation with respect to ATP synthase structure and function?
It affects ATP synthesis but not ATP hydrolysis.
The T8993G mutation results in a leucine residue at position 156 being replaced with an arginine (L156R). Molecular modelling shows the presumed location (right).
The proton flow into the matrix which drives synthesis (Panel A) is thought to be impaired.
Protons are, however, able to flow in the opposite direction when ATP is hydrolysed.
The prognosis for patients with high levels of this mutation remains poor, with death in the mid teenage years sadly typical.
Approaches targeting native ATP6 protein to the mitochondria are being pursued in the laboratory with some success (Chin et al, Cell Reports 22:2818-2826 (2018)
What are the consequences of an interruption to ATP synthesis or oxidative phosphorylation
The cell will be rapidly depleted of its ATP supply. Depending on the cell type and their metabolic requirements, death will be within a few minutes (neurons) or a few hours (muscle).
What are the common causes of failure of oxidative phosphorylation
The most common cause of a failure of oxidative phosphorylation is simply a lack of oxygen e.g. hypoxia (diminished), anoxia (total).
What is the uptake of oxygen in the mitochondria controlled by
Uptake of oxygen by mitochondria is controlled by the components of ATP production, inorganic phosphate (Pi), and ADP.
This is known as respiratory control and allows the body to adapt oxygen consumption to energy requirements (left).
Describe how cyanide and azide act as metabolic poisons
Cyanide(CN-) and azide(N3-)bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex.
This blocks the flow of electrons through the respiratory chain and consequently, the production of ATP.
Describe how malonate acts as a metabolic poison
Malonate closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. It slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate.
Describe how rotenone acts as a metabolic poison
Rotenone is a isoflavone found in the roots and seeds of some plants. It inhibits the transfer of electrons from complex I to ubiquinone.
Describe how oligomycin acts as a metabolic poison
Oligomycin is an antibiotic produced by Streptomyces that inhibits oxidative phosphorylation by binding to the ‘stalk’ of ATP synthase and blocking the flow of protons through the enzyme.
Describe how dinitrophenol acts as a metabolic poison
Dinitrophenol(DNP) is a proton ionophore which can shuttle protons across the inner mitochondrial membranes- uncouples the flow of protons with the synthesis of ATP by ATPsynthase
Can the mitochondria metabolise glucose, ADP or Pi directly
No
If a patient can respire at maximum rate all the time what does it suggest
Lack of respiratory control
If oligomycin does not stop O2 consumption, what does this suggest
protons are able to re enter the matrix independently of ATP synthase, hence ATP synthesis has been uncoupled from the proton flow.
What can DNP stimulate in healthy patients
ATPase.
What are the consequences of uncoupling the proton flow from ATP synthesis
Uncoupling of respiration from ATP synthesis leads to dissipation of the bulk of the energy within the proton motive force as heat. This explains the extreme levels of perspiration of the patient as her body tries to remain cool. It also explains her low weight despite a relatively high calorific intake.
Describe the importance of regulated uncoupling in brown adipose tissue
Regulated uncoupling of oxidative phosphorylation occurs in the brown adipose tissue of new born humans and hibernating animals.In response to a temperature drop, the release of hormones leads to the liberation of free fatty acids from triacylglycerols that in turn, activate thermogenin, also known as UCP-1.
Like DNP, UCP-1 allows protons to bypass ATP synthase thereby releasing heat from the dissipation of the proton gradient (shown below).
Explain why DNP was patented in ‘anti-fat’ medicines
DNP can induce weight loss by transporting protons across the mitochondrial membrane, thereby uncoupling oxidative phosphorylation from ATP production and markedly increasing the metabolic rate and body temperature. Subsequently DNP found its way into numerous patented anti-fat medicines for the treatment of obesity. However, the margin between the slimming dose and that required to poison or kill is slight -so slight that several patients died and many suffered permanent injury before use of the drug was abandoned in 1937.
Describe the re-oxidation of NADH and FADH2 by molecular oxygen
NADH + H+ +0.5O2 — NAD+ + H2O
FADH2 + 0.5O2 — FAD + H2O.
