All metabolic pathways share some common features and grasping the chemical logic behind them will lend to the understanding of many different pathways. If you haven’t already read my Intro into Metabolism, I highly recommend that you do (things will make a lot more sense). As a reminder, here’s the key points to think about while reading through this:
Glycolysis is a set of ten reactions in which the 6-carbon glucose molecule is broken apart and converted to two 3-carbon pyruvate molecules, making ATP and NADH along the way. By rearranging the structure of the glucose molecule, we’re able to extract useful energy for the cell. Pyruvate is a versatile molecule in that it can be used to make more glucose, generate more ATP, or be converted into useful products that the cell needs. In fact, cancer cells rely largely on pyruvate for growth and proliferation – a topic for another time. The role pyruvate plays depends on the dynamic needs of the cell and the conditions in which it finds itself. We’ll dive into the specifics of each.
Understanding glycolysis isn’t about memorizing a set of reactions. Rather, it’s much more beneficial to understand the chemical inter-conversions taking place. We want to understand the mechanism by which these reactions take place. Why is it we’re able to move a carbonyl from one carbon to another? Why does a certain alcohol get oxidized? How do we make a double bond? It’s also important to understand the energetics of these conversions. Some steps along the pathway are exergonic, some endergonic. So, how are these endergonic steps accomplished? I covered this broadly in the introductory post but here, we’ll dive into the details.
Here it is – the 10-reaction pathway that takes us from glucose to pyruvate. We can divide this pathway into two stages: 1) a preparative stage, where we set up for the extraction of energy and 2) an ATP-generation stage, or the payoff stage. Here’s the net reaction:
Glucose + 2 NAD+ + 2 ADP + 2 Pi –> 2 NADH +2 pyruvate + 2 ATP +2 H20 + 4H+
Simplistically, what’s happening is:
1) we phosphorylate glucose to trap it in the cell
2) we rearrange its carbon skeleton, converting it into a higher energy compound from which we’ll extract the energy from
3) we’ll couple the hydrolysis of those higher energy molecules to ATP synthesis
As you read through this, think of the logic behind the chemical rearranging of glucose. For example, why do we end up with pyruvate and not something else? It’s a structure that is central to many metabolic pathways, but why?
Before we get into glycolysis, it’s helpful to quickly review what nucleophiles and electrophiles are. In short, a nucleophile donates an electron pair to make a bond while an electrophile accepts them. This, along with acid-base chemistry, is at the heart of the enzymatic reactions of glycolysis. Typically, we make nucleophiles better by deprotonating them (that’s the “base” part of acid-base chemistry). A thiolate (R-S–) is a much better nucleophile than a thiol (R-S-H), for example.
Nucleophilic attack on the carbonyl carbon atom will be common (as is with many enzymatic reactions in general). We can further activate these carbonyl carbon atoms by complexing them with metal ions or making Schiff bases. An example of this is when the oxygen on the carbonyl is replaced with a nitrogen, making the carbon even more electrophilic. We’ll see all these types of reactions in glycolysis.
Step 1: Glucose to Glucose-6-Phosphate
The first reaction in the glycolytic pathway is the phosphorylation of glucose by the enzyme hexokinase. The Km of hexokinase is 0.1 mM. This is noteworthy because typical glucose concentrations at rest, in your bloodstream, are somewhere between 4-6 mM (which can obviously go up after eating). Unless you’re going into a diabetic coma, these levels never dip below 1 mM. This suggests that hexokinase is remaining fully active all the time – it’s going to be saturated at any reasonable concentration of glucose. The product of this reaction acts as negative feedback – when glucose-6-phosphate levels rise, enzyme activity drops.
Glucose, a neutral molecule, reacts with ATP and hexokinase leading to its phosphorylation on the 6 position (know your numbering – this will be important). This occurs as a random, bi-bi reaction with a negative free energy change, considered to be an irreversible step. There are two effects being accomplished here: 1) glucose is no longer neutral after this phosphorylation event – it now carries a negative charge. This is a way of trapping it in the cell, preventing it from diffusing freely out of the cell. 2) This sets the stage for the following reaction. Remember, we want to get two 3-carbon units out of the 6-carbon glucose – phosphorylation the 6 position of glucose sets up the eventual cleavage between the 3 and 4 carbons. This is the case in the muscle and other tissues. The liver is a bit different.
The liver uses glucokinase instead of hexokinase and has a Km of 5mM. This is in the range of normal glucose concentrations which means its activity is highly sensitive to the amount of glucose present. There’s a good reason for this which will become clear later on.
Hexokinase (or glucokinase) is a clamshell-looking enzyme. In its open form, the active site is exposed to solvent. Water would interfere with this reaction so when its substrates bind, hexokinase closes, excluding water from its active site. The mechanism by which glucose becomes phosphorylated is straightforward. The hydroxyl group (-OH) on the 6 position of the glucose attacks the gamma phosphate of ATP (this is the phosphate furthest from adenosine). This then cleaves the phosphoanhydride bond. Magnesium helps to orient the ATP molecule and shields the oxygen’s negative charges, enhancing the rate of reaction.
Before moving onto the next step of glycolysis, let’s discuss enolates, what they are, and what significance they hold moving forward.
The Power of the Enolate
An enolate is a deprotonated carbon, alpha to a carbonyl group.
The resonance form of this carbanion places the negative charge on the oxygen. This enolate form can be further stabilized in an enzyme’s active site through hydrogen bonding, utilizing a metal ion, or using a Shiff base discussed earlier. With a Schiff base, the negative charge of the carbanion can become formally neutral. This enolate chemistry will allow us to move carbonyls around – a common occurrence throughout the glycolytic pathway, and something we’ll see in the next step.
Step 2: Glucose-6-Phosphate to Fructose-6-Phosphate
The second step in glycolysis involves phosphoglucose isomerase, which converts glucose, a pyranose and acetal, into fructose, a furanose and ketal in a uni uni reaction.
There’s an aldehyde at the 1 position of glucose which forms a 6-membered ring with alcohol (-OH) at the 5 position. Phosphoglucose isomerase moves glucose’s carbonyl from the 1 position to the 2 position, changing it to a ketone and giving us fructose-6-phosphate, a furanose (a five-membered ring).
An acid in the active site of the enzyme protonates glucose-6-phosphate’s alcohol. This causes the molecule’s ring to open. The proton at the 2 position is then extracted by a base in the enzyme’s active site – an example of acid-base catalysis. This creates an enolate (specifically an enediol) intermediate. The electrons (now as a double bond) grab this proton back, effectively moving the carbonyl from its original place to the 2 position.
This carbonyl, being an electrophile, gets attacked by the oxygen at the 5 position, giving us a 5-membered ring. This conversion of a pyranose to a furanose sets up the eventual cleavage between the 3 and 4 carbons. There’s a very small change in free energy here, and so it is easily reversible. The position of the reaction’s equilibrium relies on flux through the system: the product concentration vs. reactant concentration.
Step 3: Fructose-6-Phosphate to Fructose-1,6,-Bisphosphate
In this step, we’re essentially making a more symmetrical-looking molecule. The goal, after all, is to produce two identical 3-carbon molecules from our original 6-carbon one. Phosphofructokinase, much like hexokinase, uses ATP to phosphorylate fructose-6-phoshphate, trapping the molecule in this furanose form. The addition of this phosphoester prevents the previous enzyme from converting this molecule back to a previous intermediate. And, as with the hexokinase reaction, this reaction is exergonic and irreversible.
This is a straightforward reaction in which the C1 of fructose-6-phosphate is phosphorylated, generating fructose-1,6,bisphosphate. Phosphofructokinase is an allosteric enzyme that serves as the key regulator of glycolysis. We’ll touch on this briefly now and explore it deeper in a separate post.
Once fructose-1,6-bisphosphate has been made, those carbons have been committed to glycolysis. It stands to reason then, that this enzyme is highly regulated. The enzyme is tetrameric and contains two different adenosine nucleotide binding sites – one in which the kinase reaction takes place and the another that’s regulatory.
It can also bind many other compounds that act as energy sensors of the cell. In a state where ATP is relatively high, the activity of phosphofructokinase is inhibited. With just a small amount of AMP present, though, the activity of this enzyme is raised substantially. Take a look at the graph below:
Again, we’ll discuss this in more depth in a separate post.
Before we look at the next step in glycolysis, let’s briefly review aldol reactions. An aldol reaction essentially makes or breaks a bond that’s beta to a carbonyl. In the example below, the hydroxyl of a beta-hydroxy ketone is deprotonated. This allows the bond between the alpha- and beta-carbons to be broken.
This gives us an enolate (which will be stabilized by a metal ion or a Schiff base in upcoming reactions which, upon protonation, produces a ketone. The other piece of the molecule (the beta-carbon) is an aldehyde. This was the whole point of moving the carbonyl from the 1 position of glucose to the 2 position of fructose that we saw earlier. We’ll see this exact type of cleavage with our next enzyme, aldolase.
Step 4: Fructose-1,6-Bisphosphate to Dihydroxyacetone Phosphate and Glyceraldehyde-3-Phosphate
Aldolase takes fructose-1,6-bisphosphate and breaks it into two 3-carbon units: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (a ketone and aldehyde, respectively).
The cleavage of fructose-1,6-bisphosphate occurs between C3 and C4 via a type 1 aldolase. Here, “type 1” (sometimes called “class 1”) refers to the enzyme using a Schiff base. Type 2 aldolases use a divalent cation like magnesium – we’ll see those eventually. Let’s look at its mechanism:
A lysine residue in the active site of the enzyme reacts with the carbonyl group of fructose-1,6-bisphosphateand forms amidium ion (a cation, or positively charged ion, formed by the addition of a proton to the nitrogen of an amide).
A base (an aspartate residue) in the active site extracts a proton from the hydroxyl group on the C4 position of the molecule. This breaks the 3-4 carbon bond, releasing our first product: glyceraldehyde-3-phosphate and creating an enamine intermediate within the enzyme
Next, the nitrogen gets reprotonated and is then hydrolyzed and releases our second product, dihydroxyacetone phosphate.
We’ve broken our original 6-carbon glucose down to two 3-carbon molecules, each of which contains a phosphate and carbonyl at the ends.
Step 5: Glyceraldehyde-3-Phosphate to Dihydroxyacetone Phosphate
Glyceraldehyde-3-phosphate and dihydroxyacetone phosphate are simply isomers (same formula, different arrangement) of one another and triose phosphate isomerase is the enzyme that equilibrates them. This interconversion involves enolate chemistry similar to what we saw with glucose-6-phosphate into fructose-6-phosphate. There is an enediol intermediate within this conversion process:
Triose phosphate isomerase is often referred to as a perfect enzyme. That is, it is only limited by diffusion – when it picks up substrate, it converts it to product. Interestingly, the Keq for this reaction lies heavily on the side of dihydroxyacetone phosphate:
Keq = [glyceraldehyde-3-phosphate]/[dihydroxyacetone phosphate] = 4.73 x 10-2
Since glyceraldehyde-3-phosphate and not dihydroxyacetone phosphate continues through glycolysis, this can seem a bit counterintuitive. An equilibrium that lies heavily on the product that does not continue forward has important implications for the pathway. Think of it as somewhat of a “molecular reserve”, providing product even under conditions where there are low levels of either of the 3-carbon molecules, always allowing us to continue onto the next step. It’s a major problem if a pathway has a middle step that “dries up” so to speak. To do so would break flux coupling and control through the pathway. As such, you can see how evolutionarily, glyceraldehyde-3-phosphate as the product to proceed forward because of this equilibrium.
This Wraps Up the Preparative Stage of Glycolysis
We’ve finished the first half of glycolysis and, in doing so, produced two 3-carbon phosphate containing molecules. These phosphates were added through the consumption of two ATP molecules. The other important point to remember is that phosphofructokinase was a key regulatory step. Next, we’ll cover the second half of glycolysis in which we gain some ATP from our initial investment and create a product that has the potential to generate much more.