The Aldolase Reaction and the Steady State

 
On banning the word "spontaneous" to describe biochemical reactions.Aldolase is an enzyme that's important in gluconeogenesis and glycolysis. I'm discussing it because RPM is describing his work on aldolase genes in Drosophila melanogaster [Aldolase in Gluconeogenesis & Glycolysis].

Fructose 1,6-bisphosphate aldolase ("aldolase") catalyzes the reaction shown below where two 3-carbon compounds are joined to produce a 6-carbon fructose molecule.


The mechanism of aldolase is described in Pushing Electrons. What I want to discuss here is the fact that this reaction is reversible. It has to operate equally efficiently in either direction.

The direction shown is part of gluconeogenesis: the synthesis of glucose. The standard Gibbs free energy change for this reaction is -28 kJ mol^-1 (ΔG°′ = -28 kJ mol^-1). This may not mean a lot to most of you but it indicates that under standard conditions the reaction gives off a lot of energy. Very negative values are associated with release of energy and energy release is favored over uptake of energy.

In terms of old fashioned biochemistry, we would have said that the reaction was spontaneous in the direction shown. In other words, the enzyme will be more likely to synthesize fructose 1,6-bisphosphate (F1,6P) than to break it down.

This perspective is very misleading since inside the cell the reaction can easily flow in either direction depending only on small changes in the concentrations of substrates and products. In the new way of looking at metabolism we no longer talk about reactions being spontaneous and we no longer use the standard Gibbs free energy changes (ΔG°′) as indicators of direction. This change in teaching was stimulated, in part, by the difficulties in explaining how the aldolase reaction could catalyze breakdown of fructose 1,6-bisphosphate to dihydroxyacetone (DAP) phosphate and glyceraldehyde 3-phosphate (G3P) in the face of a standard Gibbs free energy change that was very positive. (The value for the reverse reaction is +28 kJ mol^-1.) Those kind of reactions weren't supposed to happen in the old textbooks and it suggested that glycolysis is impossible.

Here's how we think about it today. What the standard Gibbs free energy change tells us is that under standard conditions the reaction will proceed to the right until equilibrium is reached. The standard conditions are 1M concentrations of all the substrates and products.

When enough of the substrates are converted to product the reaction will start to flow in the opposite direction until eventually an equilibrium is reached where the rate of synthesis of fructose 1,6-bisphosphate equals the rate of its breakdown. At this point the real (as opposed to standard) Gibbs free energy change will be 0 (zero). There will be no overall tendency for the reaction to flow in one direction or the other. The concentrations of substrates and products at this point will be the equilibrium values. I hope it's clear that at equilibrium the concentration of fructose 1,6-bisphosphate will be much higher than the concentrations of dihydroxyacetone and glyceraldehyde 3-phosphate. We can illustrate this in a cartoon that represents the concentrations as blobs of various sizes.


The standard Gibbs free energy change doesn't tell us whether a reaction will be spontaneous or not. Instead, it simply tells us the final concentrations of substrates and products at equilibrium. (You can calculate this using simple equations that you learn in introductory chemistry courses.) The equilibrium concentrations are the concentrations found inside the cell since almost all reactions operate at Gibbs free energy values close to zero. In other words, most biochemical reactions are near-equilibrium reactions with steady-state concentrations close to the equilibrium values.

The concentrations of the substrates and product of the aldolae reaction look like the blob cartoon shown above. If the cell is making glucose then there will be a steady trickle of substrates flowing into the reaction and this increases the substrate concentration (little blobs) a little bit so that more of it is converted to fructose 1,6-bisphosphate (F1,6P) (big blob) in order to restore the equilibrium.

Conversely, if the cell is breaking down glucose then the concentration of fructose 1,6bisphosphate will increase above the equilibrium, steady-state value and more of it will be broken down to the 3-carbon compounds. This will happen in spite of the fact that there is already a lot more F1,6-P inside the cell than G3P and DAP.

This explains why the central reactions of the gluconeogenesis/glycolysis pathways can catalyze reactions in either direction and can swich quickly from one direction to another. The key is that the steady-state concentrations inside the cell are far from the standard concentrations.