Question

Mitochondrial disease. A mutation in a mitochondrial gene encoding a component of ATP synthase has been identified. People who have this mutation suffer from muscle weakness, ataxia, and retinitis pigmentosa. A tissue biopsy was performed on each of three patients having this mutation, and submitochondrial particles were isolated that were capable of succinate-sustained ATP synthesis. First, the activity of the ATP synthase was measured on the addition of succinate and the following results were obtained. (a) What was the purpose of the addition of succinate? (b) What is the effect of the mutation on succinate-coupled ATP synthesis? Next, the ATPase activity of the enzyme was measured by incubating the submitochondrial particles with ATP in the absence of succinate. (c) Why was succinate omitted from the reaction? (d) What is the effect of the mutation on ATP hydrolysis? (e) What do these results, in conjunction with those obtained in the first experiment, tell you about the nature of the mutation?

Short answer

Succinate stimulates ATP synthesis by driving the electron transport chain. The mutation likely impairs ATP synthesis and hydrolysis, affecting overall ATP production and utilization in cells.

Step-by-step solution

Understanding the Addition of Succinate

Succinate is a substrate for the electron transport chain in mitochondria. It donates electrons, facilitating the generation of a proton gradient across the inner mitochondrial membrane, which is essential for ATP synthesis. Thus, in this experiment, succinate was added to stimulate ATP synthesis by driving the electron transport chain, leading to proton gradient formation.

Assessing the Effect of Mutation on ATP Synthesis

The mutation likely reduces the efficiency of ATP synthesis, as evidenced by muscle weakness and other symptoms. By comparing ATP synthase activity with and without the mutation in the presence of succinate, the mutation's specific impact on succinate-coupled ATP synthesis can be determined.

Reason for Succinate Omission in ATPase Activity

Succinate was omitted to focus solely on ATP hydrolysis, independent of the electron transport chain. This allows the measurement of ATPase activity without interference from the electron transport processes, isolating the enzyme's ability to hydrolyze ATP to ADP and inorganic phosphate.

Effect of Mutation on ATP Hydrolysis

The experiment without succinate aims to determine if the mutation affects the ATP hydrolysis function of ATP synthase. If ATP hydrolysis is impaired, it suggests a defect in the enzyme's ability to function properly, which would corroborate the symptoms observed.

Interpretation of Results

If the mutation affects both succinate-coupled ATP synthesis and ATP hydrolysis, it indicates a fundamental defect in ATP synthase function. This would mean the mutation disrupts both the ability to produce and hydrolyze ATP, leading to energy deficiency symptoms in patients.

Key concepts

ATP synthase

ATP synthase is a crucial enzyme found in the mitochondria, often described as an "energy converter." It plays a central role in the process of ATP production, the primary energy currency of cells. This enzyme lies at the heart of cellular respiration, facilitating the conversion of ADP and inorganic phosphate into ATP.

In a healthy state, ATP synthase harnesses the energy from a proton gradient created by the electron transport chain. As protons flow back across the mitochondrial membrane, ATP synthase uses this energy to push together ADP and phosphate into ATP. However, when a mutation occurs in any of the mitochondrial genes encoding a component of ATP synthase, as seen in some mitochondrial diseases, this process can be severely affected.

Common symptoms associated with defective ATP synthase include muscle weakness and neurological issues, due to the lack of adequate energy production in high-demand tissues like muscles and nerves. Mutations can lead to either partial or complete loss of ATP synthase function, affecting the body's energy balance.

Succinate

Succinate acts as a critical substrate in the mitochondrial electron transport chain, situated as part of the larger Krebs cycle. As it donates electrons to the chain, succinate initiates the process that leads to proton pumping and gradient creation, essential for ATP synthesis through ATP synthase.

The addition of succinate in experiments studying mitochondrial function serves a dual purpose. Firstly, it helps understand the working of the electron transport chain by supplying the necessary electrons. Secondly, it plays a role in analyzing if the ATP synthase affected by a mutation can still perform ATP synthesis coupled to this electron transport.

When assessing the impact of potential mutations, changes in succinate-driven ATP synthesis reveal how the enzyme’s efficiency may be compromised. Thus, it directly links succinate's consumption to the effective working of ATP synthase, providing insights into enzymatic mutations.

Mutation

In the context of mitochondrial disease, a mutation refers to any alteration in the DNA sequence of mitochondrial genes coding for components of enzymes like ATP synthase. Such changes in the genetic code can alter the structure and function of ATP synthase, leading to impaired energy production.

Mutations may arise spontaneously or be inherited, and they typically present with varied symptoms depending on the extent of functional loss in ATP synthase. Commonly observed symptoms include muscle fatigue, neurodegenerative disorders, and other energy-deficiency manifestations.

Studying the effects of these mutations on ATP synthase's ability to synthesize or hydrolyze ATP assists scientists in understanding the nature of the defect. Whether it affects the enzyme's function in ATP synthesis using succinate or ATP hydrolysis alone can indicate specific structural anomalies caused by the mutation.

Electron Transport Chain

The Electron Transport Chain (ETC) is a sequence of protein complexes embedded in the mitochondrial inner membrane, responsible for producing a proton gradient that powers ATP synthase. As electrons donated by molecules like succinate move through the chain, their energy is used to pump protons across the membrane, creating a potential energy differential.

This gradient is the driving force that ATP synthase uses to synthesize ATP from ADP and inorganic phosphate. Without a properly functioning ETC, ATP synthase would lack the necessary energy to produce ATP efficiently.

In cases of mitochondrial mutations affecting ATP synthase, the ETC might still perform normally, yet the final step of ATP production is compromised. This distinction helps in diagnosing specific mitochondrial defects where the proton gradient is formed, but ATP synthase cannot utilize it effectively. Understanding and examining this disconnect provides insights into the biochemical basis of mitochondrial diseases.