Hello, and welcome to Hell - or, another metabolism article. There are many articles that explain glycolysis and while I feel many of those articles do an admirable job, they fail in one key area; they fail in explaining the “why” of the matter. Specifically, they fail to explain why each step occurs the way that it does. In this article, not only will we go over every minute detail of the glycolysis pathway from chemical structure, to enzymatic reactions, but we will come to understand the why of every step until we are the most well versed on the subject. So, without further ado, I bring you glycolysis.

What is Glycolysis?

Glycolysis is the first of three major metabolic pathways that lead to the creation of energy in the form of adenosine triphosphate (ATP)[1]. No matter the circumstance, the beginning of the extraction of energy begins with glycolysis.

Where does it occur?

Glycolysis occurs in all cells [2]. More specifically, glycolysis occurs in the cytoplasm of the cell, so, outside of the organelles [3].

This is a rudimentary image of a cell (although not all cells are round). The cytoplasm is the fluid area around the various organelles.

Why does it matter?

Without glycolysis, we die. Simple enough, no? Glycolysis, because it is the critical first step to extracting energy from glucose molecules, is a step that cannot be ignored since it changes the structure of the glucose molecule in a way that makes it useable for the creation of ATP, our body’s most pure form of energy [4].

How does Glycolysis occur?

Ladies and gentlemen, rev your engines; this is the section of the article where we will examine the step by step mechanism of glycolysis and understand each step in detail. Do not worry, however, we will hold hands the entire way!

Step 1: Glucose              Glucose-6-Phosphate

Okay, so, we are currently in a position where we have a cell and we have a molecule of glucose. The molecule of glucose is outside of the cell and since we know glycolysis occurs inside of the cell, we need to transport this molecule of glucose into our cell. So, this glucose molecule diffuses across the cell membrane into the cell. However, once it is in the cell, this same glucose molecule needs to have its configuration changed (into glucose-6-phosphate) so as to prevent it from diffusing back out of the cell the same way it entered the cell [5]. Think of it as a doorway that allows you (as the glucose molecule) to enter the way you are, but once you enter, a backpack is put on you that inhibits you being able to walk through the same doorway, because your shape has changed. This change in configuration of the glucose molecule not only extinguishes the possibility for the glucose leaving the cell again, but sets up the glucose molecule for the following glycolysis steps – these are the two reasons for this first step to occur [5].

Once the glucose molecule is transported into the cell, the enzyme Hexokinase (in muscle) or Glucokinase (in liver) phosphorylate, using one adenosine triphosphate (ATP), the glucose molecule on the 6th location to create glucose-6-phosphate [6]. For Hexokinase to function, however, ATP must first interact with magnesium (Mg2+) to create a favorable configuration (MgATP) for the enzyme [8][20][21].

What is phosphorylation?

Phosphorylation is simply adding a phosphate to an organic substance. In this case, a phosphate is added to the 6th carbon of the glucose molecule, and this is the reason for the name glucose-6-phosphate.

Step 2: Glucose-6-Phosphate               Fructose-6-Phosphate

Now, we are at a point where we are located in the cytoplasm of the cell, we have a glucose-6-phosphate molecule and the body is now in a position to make a decision on what to do with this glucose-6-phosphate molecule. One option is to send it off for storage as glycogen using the metabolic process glycogenesis, if energy demands are being met and glycogen stores are capable of filling further [7]. However, if the body needs this molecule for immediate energy, then it will continue down the glycolysis metabolic pathway – we will assume this to be the case here.

Glucose-6-phosphate is then acted upon by the enzyme Phosphoglucose Isomerase that takes glucose-6-phosphate and changes its conformation to fructose-6-phosphate via isomerization [8]. It is capable of this by a three part mechanism that includes the opening of the glucose-6-phosphate ring molecule, changing the position of one of the carbons making up the ring (isomerization), and then closing the ring. This, effectively, changes the molecule from a 6 carbon ring in glucose-6-phosphate to a 5 carbon ring, as seen in fructose-6-phosphate, leading to identical chemical makeup (C6H12O6), but different structure – this is called an isomer via isomerization [8][9].

Now, it should be noted that Phosphoglucose Isomerase is a two way enzyme, which means that not only can Phosphoglucose Isomerase isomerize glucose-6-phosphate to fructose-6-phosphate, but the reverse is true, as well – fructose-6-phosphate can be isomerized back to glucose-6-phosphate [8][9]. This is controlled by the relative concentrations of both glucose-6-phosphate and fructose-6-phosphate as there is typically a low level of fructose-6-phosphate compared to glucose-6-phosphate, so if we assume Le Chatelier’s Principle that the reaction will go in favor of the lower concentration, then it will be a forward moving reaction most of the time [10]. That being the case, this step occurs in case there is an overabundance of fructose-6-phosphate and Phosphoglucose Isomerase must move in the reverse direction to covert/isomerize fructose-6-phosphate back to glucose-6-phosphate for possible storage as glycogen.


Step 3: Fructose-6-Phosphate               Fructose-1,6-Biphosphate

At this point, we have a fructose-6-phosphate molecule and a new enzyme, by the name of Phosphofructokinase, acts on the molecule to create fructose-1,6-biphosphate [6][8]. This is made possible by using another adenosine triphosphate (ATP) molecule to further phosphorylate the fructose-6-phosphate molecule on the first carbon of the now fructose ring creating fructose-1,6-biphospahte [6][8]. Phosphofructokinase is a one way enzyme so it cannot reverse the reaction [8].

The reason for this step’s existence is the fact that this is a rate-limiting step, which means it is the slowest step of the entire glycolysis metabolism pathway and dictates when glucose is further broken down beyond this point [8]. Interestingly, the relative concentration of adenosine triphosphate in the cell is the signal for furthering or stopping glucose (at this point, fructose-6-phosphate) breakdown [8]. If adenosine triphosphate (ATP) levels are low in the cell, then ATP only binds to the active site of the enzyme along with a fructose-6-phosphate molecule in preparation for phosphorylation of the 1st carbon in the ring structure [8]. However, if ATP levels in the cell are high, then ATP not only binds to the active site, but also to a regulatory site and via allosteric inhibition changes the conformation of the enzyme in a way that it no longer accepts fructose-6-phosphate for phosphorylation (this is called a “tense configuration”)[8]. Again, magnesium (Mg2+) is necessary to interact with ATP (MgATP), as with step 1 [8][20][21].


What is allosteric inhibition?

A place on an enzyme where a molecule can attach, and when this attachment occurs, it changes the shape of the enzyme to decrease its ability for accepting substrates to react together – which is what the enzyme would typically do [11].

Step 4: Fructose-1,6-Biphosphate               Dihydroxyacetone Phosphate & Glyceraldehyde-3-Phosphate

Now, we have a fructose-1,6-biphosphate molecule and hopefully you have noticed by the step title that we need to create two different molecules from this single fructose-1,6-bipshopshate molecule. This process is considerably different from the other mechanistic steps in a few key ways.

First, to do this, the body employs the service of an enzyme by the name of Fructose Biphosphate Aldolase – shortened as Aldolase [8][12]. There are two types of Aldolase enzymes, but the one found in the human body is Type I, and within the scope of Type I, there are three variations of Type I Aldolase enzymes that are separated by what type of cells of the body house them [13]. The variations are split between Aldolase A found in the muscle cells, Aldolase B is found in the liver, kidneys, stomach and intestines, and Aldolase C is found in the brain and heart; all of these Aldolase enzyme variations act in glycolysis, but only Aldolase B is also involved gluconeogenesis [13]. Now, the Aldolase enzyme acts upon fructose-1,6-biphosphate by cleaving the molecule between the 3rd and 4th carbon, effectively splitting in two [8][12].

At this point, we are dealing with dihydroacetone phosphate and glyceraldehyde-3-phosphate as our product molecules from the earlier cleavage of fructose-1,6-biphosphate by Aldolase [8]. However, only glyceraldehyde-3-phosphate is usable for continuation down the glycolysis pathway as future enzymes are structured in a way that they only react with this configuration [12]. Because later enzymes only react with a specific conformation of 3 carbon atoms, 7 hydrogen atoms, 6 oxygen atoms, and 1 phosphate (C3H7O6P) making up glyceraldehyde-3-phosphate, this is the reason for the specific cleavage on the bond between the 3rd and 4th carbon; due to this tactical separation, the body is then able to take the assumed “byproduct”, dihydroxyacetone phosphate (also C3H7O6P), and reconfigure it via a separate reaction, to fit the C3H7O6P configuration desired by later enzymes [12].


Step 4B: Dihydroxyacetone Phosphate               Glyceraldehyde-3-Phosphate

This side step of reorganizing the isomer dihydroxyacetone phosphate (C3H7O6P) to the glycolysis favored isomer glyceraldehyde-3-phosphate (C3H7O6P) is accomplished using the enzyme Triose Phosphate Isomerase [8][12]. Glycolysis continues with the original glyceraldehyde-3-phosphate while this step occurs, and once this step is finished, the newly isomerized glyceraldehyde-3-phosphate is then also carried through glycolysis [12]. This strategic cleavage not only supplies the later enzymes the ability to react with the appropriate molecule isomer, but allows one original glucose molecule to offer two molecular units to go through glycolysis as opposed to just one.

Step 5: Glyceraldehyde-3-Phosphate               1,3-Biphosphoglycerate

At this point of the glycolysis pathway, we have a glyceraldehyde-3-phosphate (technically, two, just at different points in time) and we use the enzyme Glyceraldehyde-3-Phosphate  Dehydrogenase, along with some other substituents to synthesize 1,3-biphosphoglycerate [8]. This step sets up the following step, which we will see produces our first unit of energy.

Using a glyceraldehyde-3-phosphate molecule, Glyceraldehyde-3-Phosphate Dehydrogenase combines oxidized nicotinamide adenine dinucleotide (NAD+) and an inorganic phosphate (Pi) to create 1,3-biphosphoglycerate [8]. This is done with the NAD+ being reduced with the addition of a hydrogen atom (NADH) taken from the glyceraldehyde-3-phosphate molecule [8]. Then, an inorganic phosphate attaches, changing the conformation of the molecule in favor of the following step – this biphosphate molecule is now, not only in the correct conformation to follow through glycolysis, but in a high energy state prepared to surrender a phosphate group – as we will see shortly [8].


What is oxidized nicotinamide adenine dinucleotide (NAD+)?

Nicotinamide adenine dinucleotide is a nitrogenous based molecule that is synthesized within the body and can also be created via the digestion and absorption of vitamin B3/Niacin [14]. This molecule, or coenzyme (as it is often needed in furthering reactions), can come in various forms, but in this instance, it is oxidized, meaning it has lost an electron and is receptive to gaining an electron back [15]. In this step, NAD+ denotes oxidation, so the addition of a hydrogen fulfills the need for an electron (as the hydrogen has an electron), and from a biochemical stand point, this allows glyceraldehyde-3-phosphate to have its conformation changed, as well as open the door for an inorganic phosphate to attach and finish the synthesis of 1,3-biphosphoglycerate. Variations of NAD are found throughout the cell and critical to glycolysis, the Krebs’ Cycle, and the electron transport chain [14].

What is inorganic phosphate?

Inorganic phosphate is an unattached phosphate, or free phosphate, that is specifically not associated to an organic compound. Inorganic phosphates are also found in the cell, waiting to be used by various mechanisms [16]. In this case, the inorganic phosphate allows the enzyme to synthesize a high energy molecule via its addition.

Step 6: 1,3-Biphosphoglycerate                 3-Phosphoglycerate

Currently, we have a molecule with two phosphate groups creating a high energy yield, and this step takes care of creating some energy from that high energy molecule [8]. This is made possible using the enzyme Phosphoglycerate Kinase as it removes the phosphate off the 1st carbon and is attached to an adenosine diphosphate (ADP) molecule to create adenosine triphosphate (ATP), our most basic energy molecule used in life [8][12]. Again, because of the ATP present, magnesium (Mg2+) is also present in the reaction [20][21].

Step 7: 3-Phosphoglycerate                2-Phosphoglycerate  

The aim of this step is to reconfigure the molecule with the same chemical makeup; so, we are effectively isomerizing it like we have in some previous steps (Step 4B, for example)[8]. This new isomer allows the final reactions of glycolysis to occur. The enzyme Phosphoglycerate Mutase attaches to 3-phosphoglycerate, but instead of simply moving the phosphate from 3-phosphoglycerate to the 2nd carbon, the enzyme phosphorylates itself, then takes a phosphate from a phosphorylated amino acid and attaches that phosphate group to the phosphoglycerate’s 2nd carbon to synthesize 2-phsophoglycerate [8][17].


Step 8: 2-Phosphoglycerate               Phosphoenolpyruvate

With 2-phosphoglycerate, the cell is now in a position to do two things in this one step. Not only is the final molecule of glycolysis formed in this step, but water is also produced – maximizing the use of the 2-phosphoglycate molecule [12].

A magnesium ion (Mg2+) interacts with the first activation site on the Enolase enzyme to change its conformation in a way that allows the molecule to attach, and then a second magnesium ion interacts with the second activation site of the enzyme to continue the catalytic reaction of 2-phosphoglycerate [8][18]. This catalytic reaction causes dehydration by removing a proton (H+) and an OH- group to form water (H2O) [12]. Not only that, a change in bond strength between the 2nd and 3rd carbons make the molecule less stable, but more energetically viable [12]. These two occurrences are the reason for this step, as well as the product of phosphoenolpyruvate.


Step 9: Phosphoenolpyruvate                Pyruvate

This is the final step of the glycolysis process and involves the removing of the final phosphate group from phosphoenolpyruvate to be added to an adenosine diphosphate (ADP) to synthesize the final adenosine triphosphate (ATP) of the glycolysis process with an end product of pyruvate. This step is made possible by the enzyme Pyruvate Kinase once potassium (K+)  and magnesium (Mg2+)  attach to it [8][19][21].

This step is a regulatory step like steps 1 and 4 in the liver as Pyruvate Kinase is synthesized more readily when high glucose levels in the cell are sensed [8].



In total, we begin with a single D-glucose molecule, we use 2 ATP to phosphorylate it and make it energetically viable, then we split the 6 carbon glucose molecule into two 3 carbon molecules, then using these two 3 carbon molecules we gather 1 NADH per molecule for a total of 2 NADH molecules, and 2 ATP per molecule for a gain of 4 ATP, and each molecule is transformed into a pyruvate molecule with a net result of:

2 Pyruvate molecules

What happens after Glycolysis?

At this point, it is highly dependent on the physiological state of the cell. If it is receiving sufficient oxygen, the body continues with aerobic metabolism via the Krebs’ Cycle (also named the TCA Cycle) to produce much more energy; the body is typically in this state, so this is most common [20]. On the other hand, if the body is physiologically stressed to a point that it is not intaking and distributing oxygen rapidly enough for the cells to use aerobic metabolism, then the cell goes into anaerobic metabolism via Fermentation to recycle NADH obtained in glycolysis (as detailed above) to create a bit more ATP at a more immediate rate [20]. These are the reasons for the terms aerobic and anaerobic glycolysis.


Writer: Nicolas Verhoeven

Note: Source #2 and #4 are the same.

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[14] Sauve, A. A. (2007). NAD+ and Vitamin B3: From Metabolism to Therapies. Journal of Pharmacology and Experimental Therapeutics, 324(3), 883-893. Retrieved from

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[16] Bergwitz, C. (2011). Phosphate sensing. Advanced Chronic Kidney Disease, 18(2), 132-144. Retrieved from

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