Control of cellular respiration

Eukaryotic aerobic cellular respiration is the process of producing ATP by capturing energy that is released when food molecules are oxidized, with mitochondria playing a central role and oxygen acting as the final oxidizer. This is one of the core topics of Bio 6B. We'll go into this in detail, with explanations from the textbook, lecture videos, and other sources. I've created several pages related to cellular respiration:

Cells need to make enough ATP to meet their energy needs, but it's important to slow OXPHOS down when ATP isn't being used rapidly. Not only is excess catabolism wasteful, but it also generates dangerous reactive oxygen species (ROS). Cells need to adjust their rates of cellular respiration from moment to moment to maintain an appropriate energy balance. There are several mechanisms for this.

Feedback inhibition regulates glycolysis

Cellular respiration is a multi-step process, including glycolysis, the citric acid cycle, and oxidative phosphorylation. Like many biochemical pathways, it's controlled in part by feedback inhibition: ATP, the end product of OXPHOS, inhibits the beginning of glycolysis.

PFK is allosterically inhibited by ATP

When ATP builds up in a cell, it inhibits phosphofructokinase (PFK), an enzyme that catalyzes an early step in the energy investment phase of glycolysis.

Allosteric inhibition of PFK by ATP.

As a kinase, PFK uses ATP as a substrate to phosphorylate a sugar molecule. Thus PFK has an active site for ATP. In addition, the enzyme also has a separate allosteric site for ATP.

When ATP concentration is low, PFK uses the available ATP to phosphorylate sugar and get glycolysis going. As ATP concentration increases, ATP binds to the allosteric site, inhibiting the enzyme's activity.

Like many biochemical pathways, glycolysis is controlled by allosteric regulation of an enzyme near the beginning of the pathway. Once PFK is inhibited, all the subsequent steps will also be slowed.

You might also wonder why hexokinase, the first enzyme in glycolysis, isn't the target of allosteric regulation rather than PFK. There are two reasons why this wouldn't make sense:

Much of the carbohydrate you catabolize comes from your body's stores of glycogen (a polymer of glucose that is stored in the liver and muscle). When glycogen is broken down (by the enzyme glycogen phosphorylase), the glucose monomers are released with a phosphate already attached, so they skip the hexokinase step of glycolysis.

When free glucose enters cells from the bloodstream, it gets phosphorylated by hexokinase, which traps the glucose-6-phosphate inside the cell. This useful reaction continues even when the rest of glycolysis is inhibited. The glucose-6-phosphate can be used in other pathways aside from glycolysis.

If you're extremely curious, you might ask why it's not the second enzyme, phosphoglucoisomerase, that gets allosterically regulated. That enzyme also functions in another pathway, gluconeogenesis, that is the opposite of glycolysis. Both processes would be inhibited if that enzyme was inhibited. PFK is the first enzyme in glycolysis that's always necessary and is unique to glycolysis.

PFK is allosterically activated by AMP

Adenosine monophosphate, or AMP, accumulates when ATP level runs low. Increasing AMP level acts as a signal to increase the activity of PFK.

Allosteric activation of PFK by AMP.

Here's how that works:

When a cell is amply supplied with energy, its ATP concentration will be high; as you've seen, this inhibits PFK. On the other hand, when energy runs low, ATP gets converted to ADP by cellular work. Some of the ADP gets converted back to ATP and AMP in this reaction (catalyzed by adenlyate kinase):

ADP + ADP ↔ ATP + AMP

The ΔG for this reaction is close to zero. If it was left alone, this reaction would reach an equilibrium with equal concentrations on both sides (or 2x [ADP] = [ATP] + [AMP]). However, in a living cell, there's always something pushing it away from equilibrium. When ATP gets used up, the adenylate kinase reaction gets pushed toward the right to compensate, resulting in an increase in AMP concentration. Thus, rising AMP concentration signals a need for energy. AMP binds to its own allosteric site on PFK, pushing the enzyme toward its active configuration and speeding up glycolysis.

PFK is allosterically inhibited by citrate

There is one more allosteric regulator of PFK:

Allosteric activation of PFK by ATP, AMP & citrate.

Citrate binds to yet another allosteric site, pushing PFK toward its inactive configuration.

Citrate is one of the intermediates in the citric acid cycle (which is why it's called the citric acid cycle). Citrate is produced in the mitochondrial matrix, but it can be transported out to the cytosol. Increasing cytosolic citrate concentration may indicate that glycolysis is feeding carbons to the citric acid cycle faster than they can be oxidized to feed the electron transport chain, so it makes sense to slow glycolysis down when citrate builds up. (In fact, a buildup of citrate may be correlated with an increase in ROS formation.) Citrate is also used as a starting molecule for several other pathways, so its metabolism is complex.

Upregulation and downregulation

As you've seen, PFK is allosterically regulated in several different ways. It doesn't make sense to think of this enzyme simply being "on" or "off." Each cell has numerous copies of PFK, and each copy is continually being subjected to competing allosteric activators and inhibitors. The end result is that the cell subtly upregulates an downregulates its rate of catabolism to meet its ever-changing needs.

Phosphofructokinase is not the only regulation point for cellular respiration. Keep in mind that cellular respiration isn't only a set of isolated catabolic processes; many of the intermediates can also be used as starting materials for various anabolic processes. In this complex web of pathways, many other enzymes are regulated, controlling various tradeoffs between catabolism and anabolism.

Review

Terms & concepts

  • Active sites vs. allosteric sites
  • Allosteric regulation
  • AMP, ADP, ATP: how they are converted.
  • Citrate.
  • Feedback inhibition
  • Kinases in glycolysis
  • Phosphofructokinase

Review questions

  1. Explain the roles of ATP, ADP, and citrate in regulating the activity of PFK. In what way do these molecules provide information about the cell's energetic state?
  2. Phosphofructokinase (PFK) is the main point at which the glycolysis pathway is regulated. Why is this enzyme  the one that primarily controls glycolysis, rather than another enzyme earlier or later in the pathway?
  3. Where is citrate produced? How does it influence glycolysis?

References & further reading

Videos

Allosteric Regulation of Phosphofructokinase I. Laura Slusser. This 3-minute video illustrates the basics of PFK regulation.

Reading

Phosphofructokinase (Proteopedia). In addition to a spinning, animated PFK picture, this site giives some information on how the enzyme is regulated.

Biochemistry by Berg, Tymoczko, and Stryer. This classic (and continually updated) textbook is my best source for understanding biochemical concepts. The chapter on glycolysis gives a clear account of how the process is regulated.

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