Question

An investigator is measuring the activity of various enzymes involved in reactions of intermediary metabolism. One of the enzymes has greatly decreased activity compared to reference values. The buffer of the assay contains citrate. Which of the following enzymes will most likely be directly affected by the use of citrate? (A) Fructose-2,6-bisphosphatase (B) Isocitrate dehydrogenase (C) Phosphofructokinase-1 (D) Pyruvate carboxylase

Short answer

Phosphofructokinase-1 (C).

Step-by-step solution

Identify the Context of the Problem

The problem involves the inhibition of an enzyme due to the presence of citrate in the assay buffer.

Understand the Role of Citrate

Citrate is a key molecule in metabolism that can inhibit certain enzymes, specifically those involved in glycolysis.

Evaluate Each Enzyme with Respect to Citrate

Assess if each enzyme listed is involved in glycolysis or the TCA cycle, and if its activity is influenced by citrate.

Enzyme A: Fructose-2,6-bisphosphatase

Fructose-2,6-bisphosphatase is involved in the regulation of glycolysis and gluconeogenesis, but citrate does not directly inhibit its activity.

Enzyme B: Isocitrate Dehydrogenase

Isocitrate dehydrogenase is a key enzyme in the TCA cycle. Citrate does not directly inhibit its activity.

Enzyme C: Phosphofructokinase-1

Phosphofructokinase-1 (PFK-1) is a rate-limiting enzyme in glycolysis and is known to be directly inhibited by citrate.

Enzyme D: Pyruvate Carboxylase

Pyruvate carboxylase is involved in gluconeogenesis, not glycolysis, and citrate does not directly inhibit its activity.

Conclusion

Based on the steps above, Phosphofructokinase-1 (PFK-1) is the enzyme most likely directly affected by citrate.

Key concepts

Glycolysis

Glycolysis is a crucial pathway in cellular metabolism that breaks down glucose to produce energy. It occurs in the cytoplasm of cells. The process converts one molecule of glucose into two molecules of pyruvate, generating a net gain of two ATP molecules and two NADH molecules.
Glycolysis consists of ten steps, divided into two phases: the energy investment phase and the energy payoff phase:

• Energy Investment Phase (first 5 steps): Uses 2 ATP molecules to phosphorylate glucose and its intermediates.
• Energy Payoff Phase (last 5 steps): Produces 4 ATP and 2 NADH molecules by substrate-level phosphorylation.

Glycolysis is anaerobic; it doesn't require oxygen. It's essential for providing energy in a variety of cell types, especially during intense exercise or in tissues that lack sufficient oxygen supply, such as red blood cells.
Glycolysis also provides intermediates for other pathways, including the TCA cycle (Krebs cycle) and fermentation.

Phosphofructokinase-1

Phosphofructokinase-1 (PFK-1) plays a pivotal role in regulating glycolysis. It catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, using ATP.
PFK-1 is a rate-limiting enzyme, meaning it controls the pace of glycolysis. The activity of PFK-1 is regulated by several molecules:

• Activators: AMP, ADP, and fructose-2,6-bisphosphate.
• Inhibitors: ATP and citrate.

When citrate levels are high, it signals that there's abundant energy available. Consequently, citrate binds to PFK-1 and reduces its activity, slowing down glycolysis.
This feedback mechanism ensures that glycolysis proceeds efficiently, helping maintain cellular energy balance.

TCA Cycle

The Tricarboxylic Acid (TCA) cycle, also known as the Krebs cycle or citric acid cycle, occurs in the mitochondria. This cycle is crucial for aerobic respiration and energy production.
The TCA cycle involves a series of chemical reactions that generate high-energy molecules, such as NADH and FADH₂, from acetyl-CoA derived from carbohydrates, fats, and proteins.
The main products of the TCA cycle are:

• 3 NADH
• 1 FADH₂
• 1 GTP (which can convert to ATP)
• 2 CO₂

The high-energy molecules produced in the TCA cycle feed into the electron transport chain, driving the production of large amounts of ATP through oxidative phosphorylation.
Enzymes within the TCA cycle are regulated by substrates and feedback inhibition to ensure efficient energy production and maintain cellular homeostasis.