Enolase

Enolase is an enzyme, more particularly, an active metalloenzyme. This enzyme belongs to the family of lyases, the hydro-lyases, breaking the carbon-oxygen bonds, and is present in all tissues and organisms involved in glycolysis or fermentation. The optimal pH of this enzyme is 6.5 in humans.Its main function is to intervene in the 9th step of glycolysis (the penultimate step in this metabolic pathway), a step in which occurs the dehydration of 2-phosphoglycerate (2-PG) in phosphoenolpyruvate (PEP), a product that will be used in the next and final step for the production of energy (ATP).
Enolase has three different isoforms: the ENO1 or alpha-enolase (in muscle tissue); ENO2 or gamma-enolase or neuro specific enolase (in neurons); ENO3 or beta-enolase (in skeletal muscle cells). Enolase has a molecular weight of about 100000 Daltons (depending on the isoform). In humans, the α-enolase has two antiparallel subunits, which have two domains that establish hydrophobic interactions. The subunits interact via salt bridges, involving arginine and glutamate.
The specific enolase to neurons is released in a wide variety of diseases, such as multiple sclerosis or stroke, or myocardial infarction.
In several medical experiments, it was employed enolase concentrations in samples in an attempt to diagnose certain conditions and its severity. Several studies demonstrated that different levels of enolase may also be associated with tumor growth or with the occurrence of myocardial infarction or stroke, so it was inferred that the levels of enolase serve as an indicative of the prognostic evaluation of victims of cardiac arrest.
Enolase inhibitors have been utilized in health care for the treatment and prevention various diseases, such as anti-trypanosome drugs and more recently as anticancer agents. Enolase can be inhibited by fluoride ion (F-). The fluoride forms a complex with magnesium and phosphate, which binds to the active center of the enzyme rather than the substrate 2-PG, preventing the conversion of 2-PG into PEP, decreasing the production of PEP and, consequently, ATP.
Intake of fluoride-containing water inhibits the catalytic activity of enolase of bacteria present in oral cavity (highly dependent on glycolysis due to the anaerobic environment), interrupting glycolysis and, thus, bacterial fermentation (decreased acid production), preventing the formation of dental caries.

Text written by:
Inês Carvalho
Junjie Lin
Maria Alves
Susana Pinto
.

Video with a rap about glycolysis

Music about glycolysis

This song of Dr. Ahern is about glycolysis. It was made from the music God Rest Ye Merry Gentlemen and is a very funny way to know the 10 steps of glycolysis! :)

Download the music here

Test Gently on Glycolysis

In glycolysis, a glucose ring's first turned to G6P
The enzyme hexokinase adds a phosphate – PO3
The glucose then turns fructose – 6 carbons, called F6P
Phosphoglu-cose i-som-er-ase, you see
Makes F6P
Phosphoglu-cose i-som-er-ase, you see

Fructose-1,6-bisphosphate, also known as FBP
By phosphofructokinase has a second PO3
By aldolase, it's cleaved in 2, one half gives GAP
Glyceraldehyde-3-phosphate: GAP
It goes on, you see
Through glycolysis, this lucky GAP

The second half, DHAP, can’t carry on this way
You need to change this dihydroxyacetone phosphate
To GAP, so call in "TIM", he’ll make things go his way
Triose phosphate isomerase – the same
TIM is his name TPI and TIM, they are the same

Glyceraldehyde-3-phosphate de-hy-dro-ge-nase is next
1,3-biphosphoglycerate is made; it is the best
A reaction of high energy – it says so in my text
GAPs go to 1,3-BPG
Add PO3
GAPs go to 1,3-BPG

The PO3's then lost, 'cuz phosphoglycerate kinase
By using ADP, it makes 3-phosphogly-cer-ate
This turns to 2PG with phosphoglycerate mutase
Losing water when it meets enolase
E-no-lase
Losing water when it meets enolase
Too many steps, I’m kinda lost, so let me get this straight –
A phosphate and an OH group switch places in step eight
Hence "mutase", 'cuz it changed, but what the heck is
enolase?
It makes phos-pho-enolpyruvate
Isn’t it great?
It makes phos-pho-enolpyruvate

In the 10th and final step (Hooray!) we make our pyruvate
Pyruvate kinase is our friend, he takes us all the way
The phosphate and the double bond – please take them both
away
Leaving only our precious pyruvate
Py-ru-vate
Glycolysis is done, oh happy day!
.

Music about glycolysis (2)

Dr. Ahern (www.davincipress.com/metabmelodies.html) has based on the song A Few of My Favorite Things to dedicate a music to glycolysis. Here it is the link for the download.

http://www.mediafire.com/?9p72whbfjhfctxp

Instructor sings

Aldehyde sugars are always aldoses and

If there's a ketone we call them ketoses

Some will form structures in circular rings

Saccharides do some incredible things

Onto a glucose we add a 'P' to it

ATP energy ought to renew it

Quick rearranging creates F6P

Without requiring input energy

Everyone Sings

At a high rate

Add a phosphate

With PFK

F1,6BP is made up this way

So we can run and play

Instructor sings

Aldolase breaks it and then it releases

DHAP and a few G3Pieces

These both turn in to 1,3 BPG

Adding electrons onto NAD

Phosphate plus ADP makes ATP

While giving cells what they need - en-er-gy

Making triphosphate's a situa-shun

Of substrate level phosphoryla-shun

3-B-P-G

2-B-P-G

Lose a water

PEP gets a high energy state

Just to make py-ru-vate

Instructor sings

So all the glucose gets broken and bent

If there's no oxygen cells must ferment

Pyruvate / lactate our cells hit the wall

Some lucky yeast get to make ethanol

This is the end of your glucose's song

Unless you goof up and get it all wrong

Break it, don't make it to yield ATP

You'll save your cells from fu-til-i-ty

Everyone Sings

The Sound of Glucose

Utilization of different monosaccharides on glycolysis

There is nothing new in the fact that glycolysis serves to degrade glucose, the most abundant monosaccharide in our diet. But what happens to other monosaccharides we eat? Can we degrade them for energy? If so, can we rely on glycolysis for this?
The answer to both questions is yes. Indeed, glycolysis allows the degradation not only of glucose but also of other monosaccharides such as fructose, galactose and mannose. For that, these monosaccharides have to be converted into glycolytic intermediates.
FructoseFructose is obtained from the diet in foods such as fruits, honey and cereals, for example.

 

In extrahepatic tissues, which lack the enzyme glucocinase, fructose is converted to fructose-6-phosphate by hexokinase, the first enzyme of glycolysis. It should be noted that the hexokinase uses glucose as preferred substrate, but can also phosphorylate other monosaccharides such as fructose, for example. Since fructose-6-phosphate is an intermediate of glycolysis, it can then follow the normal glycolytic pathway.

Alternatively, in liver, fructose has to undergo a different process, because the glucocinase is very specific for glucose, failing therefore to phosphorylate fructose. In this case, fructose is phosphorylated by frutocinase, producing fructose-1-phosphate. Thereafter, the aldolase, which is the 4^th enzyme of glycolysis, acts on fructose-1-phosphate, cleaving it to dihydroxyacetone phosphate and glyceraldehyde. The former molecule is already an intermediate of glycolysis, so it can proceed in that pathway, but not the latter. Therefore, the glyceraldehyde is subsequently phosphorylated by glyceraldehyde kinase, leading to glyceraldehyde-3-phosphate, which is also a glycolytic intermediate.
GalactoseGalactose is found mainly in dairy products because it is a component of lactose.

  

 

Galactose is first phosphorylated to galactose-1-phosphate by galactocinase. Then galactose-1-phosphate will receive an UDP molecule, resulting in UDP-galactose. This reaction is catalyzed by galactose-1-phosphate uridyl transferase. UDP-galactose is then epimerized to UDP-glucose, by the action of UDP-galactose-4-epimerase. The UDP-glucose undergoes an exchange of its UDP portion by a phosphoryl group, producing glucose-1-phosphate, which is then converted to glucose-6-phosphate (glycolytic intermediate) by the action of phosphoglucomutase (this enzyme also participates in the metabolism of glycogen...).

Mannose
Mannose can be found, for example, in beans, peas, or similar vegetables.

Mannose is phosphorylated by hexokinase originating mannose-6-phosphate. The mannose-6-phosphate is converted to fructose-6-phosphate (a glycolytic intermediate) by fosfomanose isomerase.

Main bibliographic sources:

- Quintas A, Freire AP, Halpern MJ, Bioquímica - Organização Molecular da Vida, Lidel

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers

Regulation of glycolysis

Glycolysis provides different cell molecules important for cellular proper functioning. The main donor of chemical energy in most of our processes is ATP. However, it also provides precursors for many other processes such as synthesis of amino acids, for example. Thus, it is essential to have a strict regulation of glycolysis, so that the cell can respond to different needs of ATP or other metabolites.

During his studies on fermentation of glucose by yeast, Louis Pasteur discovered that the rate and amount of glucose consumed was higher in anaerobic than in aerobic conditions! At first glance this may seem strange, because aerobic metabolism is normally associated with something more advantageous for the cell. In fact, the biochemical explanation is simple: under anaerobic conditions one molecule of glucose generates 2 molecules of ATP, but under aerobic conditions generates 30 or 32 molecules of ATP. Simplifying, if one thinks that in five minutes the yeast will need to get 30 ATP, this means that under aerobic conditions it will only need to spend a molecule of glucose in that time, while under anaerobic conditions, as the process is less profitable in the energy point of view, it is necessary to spend 15 molecules of glucose. That is, the flow of glucose through the glycolytic pathway is regulated depending on cell ATP levels (as well as adequate supplies of glycolytic intermediates to biosynthetic roles).

As I have mentioned in previous posts, glycolysis has 10 reactions, and there are three regulatory points (irreversible reactions). The enzymes that catalyze them are the hexokinase (reaction 1), PFK-1 (reaction 3) and pyruvate kinase (reaction 10). As these reactions are the limiting steps of glycolysis, changes in speed of action of their enzymes will alter the overall speed of the glycolytic pathway.

Of the three regulatory enzymes, the main one is PFK-1. This may seem strange, because indeed the most logical situation was that the main regulatory enzyme was the first ... Again, there is a very simple explanation for this. What is happening is that hexokinase is an enzyme also common to other metabolic processes (synthesis of glycogen and pentose phosphate pathway). In other words, despite being a regulatory enzyme, it is not unique to glycolysis. Thus, the main point of regulation of glycolysis has to be the second, ie, the reaction catalyzed by PFK-1.

One thing I usually tell to my students is that in this part of metabolic regulation is preferable to understand why certain molecules activate and inhibit some pathways, thus avoiding to memorize endless lists of modulators... Of course it is not always possible to make a direct argument for understand why some molecules act as activators and others as inhibitors, but whenever possible I will develop this idea... Let's move on to a list of the main modulators of glycolysis.

Activators of hexokinase:
- Fructose-1-phosphate (liver) – It competes with fructose-6-phosphate to the regulatory protein of glucocinase, canceling its inhibitory effect.
- Inorganic phosphate (Pi) – It is a player in the glycolytic process (involved in reaction 6) so it makes sense that if it has a regulatory role, is a stimulating one.
Inhibitors of hexokinase:
- Glucose-6-phosphate (muscle) - It makes sense that functions as an inhibitor because it is the reaction product. If we have too much product, we will not need to continue to produce more ...
- Fructose-6-phosphate (liver) – It is the product of the following reaction (reaction 2), but can be interpreted the same way as the molecule before. That is, if we are to accumulate the intermediate formed from the reaction product, there is no point in continuing to make more product. This inhibition occurs through a protein called regulator protein of glucocinase.

Activators of PFK-1:
- Fructose-2,6-bisphosphate (liver) – It is the most significant allosteric regulator of PFK-1, reducing its affinity for the inhibitors ATP and citrate. It is produced in response to insulin and degraded in response to glucagon.
- Fructose-6-phosphate – It is the substrate, so it makes sense that if we have much substrate the enzyme is activated.
- ADP and AMP – They are produced when ATP is spent, thus indicating a low energy state. Therefore, it makes perfect sense that they can activate glycolysis, so that the cell can replenish their normal energy values. They activate the enzyme because they relieve the inhibition caused by ATP.Inhibitors of PFK-1:
- Glucagon (liver) - This hormone is produced in a state of hypoglycemia and aims to raise the concentration of glucose in the blood. So it makes perfect sense that it inhibits glycolysis, because this process consumes glucose, which will further accentuate the reduced blood glucose concentration. As mentioned earlier, the glucagon decreases the levels of fructose-2,6-bisphosphate
- ATP - The main objective of glycolysis is to produce energy (ATP). So if the cell already has ATP, it makes sense that glycolysis is inhibited, thus preventing an unnecessary waste of a precious metabolic fuel as glucose! ATP inhibits PFK-1 because it decreases the affinity of the enzyme for its substrate, fructose-6-phosphate.
- Citrate – It stresses the inhibitory effect of ATP. This molecule is the first intermediate of the following step of aerobic catabolism, the Krebs cycle. So if we are accumulating Krebs cycle intermediates, it is useless to continue to perform glycolysis.
- Phosphoenolpyruvate – It is an intermediate of glycolysis that is formed in the penultimate reaction. If there is an accumulation of this intermediate, the reactions above have to be inhibited in order to prevent a further accumulation of the molecule.
- H⁺ - This enzyme is particularly sensitive to changes in pH, functioning as a "switch" that turns off, for example, when we make an exaggerated lactic fermentation (produces H⁺), preventing an even greater acidification.

Activators of pyruvate kinase:
- ADP - The reason is the same as mentioned above for the PFK-1, ie, is an indicator of an energy deficit, so it will lead to an activation of glycolysis.
- Fructose 1,6-bisphosphate - an intermediate of glycolysis that is formed in a reaction prior to the one catalyzed by pyruvate kinase. So if we are accumulating an intermediate produced in an earlier stage, we have to activate this enzyme in order to counteract this accumulation (as when a dam is accumulating too much water, and to restore normal values ​​is necessary to open the gate ...).
- Dephosphorylation (liver) - Induced, for example, by insulin, which makes sense, given that insulin is produced in a situation of excess blood sugar (hyperglycemia) and will activate the process (one of them is glycolysis!) that consume glucose in order to lower the blood glucose concentration.
Inhibitors of pyruvate kinase:
- ATP – It is a carrier of chemical energy and one of the end products of glycolysis, so there is no need to continue the breakdown of glucose. It decreases the affinity of the enzyme for phosphoenolpyruvate.
- Acetyl-CoA – It is the molecule in which the product of this reaction (pyruvate) is converted in the case of aerobic catabolism. Therefore, if acetyl-CoA accumulates, it makes no sense to continue to synthesize pyruvate, so the enzyme is inhibited.
- Long-chain fatty acids.
- Phosphorylation (liver) - Induced, for example, by the action of glucagon, which, as mentioned earlier, will have as main function to raise blood glucose levels. To this end, it inhibits, for example, glycolysis.
- NADH - as we shall see in more detail when I speak of cellular respiration, NADH has potential to create molecules of ATP, which signals a high energy state of the cell. In this situation, it is not necessary to resort to glycolysis for more energy.
- Alanine - This amino acid (one of the 20 standard amino acids) can lead to pyruvate (the reaction product of pyruvate kinase!), by removal of its amino group. So if there is a molecule that can directly lead to pyruvate, we do not need to spend more glucose.

In short, we can make some generalizations about the regulation of metabolic pathways, which will be useful to understand the regulation of other processes.
First, energy molecules such as ATP, or potential energy, such as NADH are, in general, inhibitors of catabolism. This is very easy to understand if we think that the main objective of the catabolism is to produce energy. If the cell already has this ability it does not need to degrade more nutrients to produce energy! The oppposite reasoning applies to ADP, AMP and NAD⁺, because any one of these molecules indicates an energy deficit in the cell (remember that when we spend ATP we obtain ADP or AMP, and when we spend NADH we obtain NAD⁺...) so it will be necessary to restore energy levels, and this activates the catabolism.
Second, the reaction product or intermediates formed from this (products of reactions following the reaction we are considering) are inhibitors. On the other hand, the substrate, or intermediaries which will originate the substrate (formed in reactions prior to the reaction that we are considering) are activators.

Main bibliographic sources:

- Quintas A, Freire AP, Halpern MJ, Bioquímica - Organização Molecular da Vida, Lidel

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers

Glycolysis (enzymes of the payoff phase)

The payoff phase, as I mentioned earlier, concerns the whole of the last five reactions of glycolysis and allows the cell to obtain energy in this process. Here are some ideas on the enzymes of phase 5 payoff ...
6^th enzyme – Glyceraldehyde-3-phosphate dehydrogenase

This enzyme, often abbreviated to GAPDH, is presented in the form of a tetramer. Each subunit has about 35.9 kDa (331 amino acids) and shows how a molecule of cofactor NAD+. The subunits are designated by O, P, Q and R and are independent of each other. That is, each subunit catalyses the reaction without the intervention of others. As described in the post about the reactions of the payoff phase, the reaction catalyzed by this enzyme is a double one, involving an oxidation and an addition of a phosphate group. It is an enzyme that may be affected by the presence of arsenic in the body, causing the yield of glycolysis to become null. Its mechanism of action involves both a covalent catalysis and acid-base. To do this, it is essential the participation of cysteine ​​149 and histidine 176 for both types of catalysis, resectivamente. The substrate binds covalently to cysteine​​, forming a hemitioacetal. The laboratory level this enzyme is widely used (I also use ...) as a positive control techniques such as immunoblotting or RT-PCR, because in general their expression is constant in almost all cell types. So it is possible to determine changes in the expression of a certain gene or in the presence of a given protein by comparing it with the levels of GAPDH.

7^th enzyme – Phosphoglycerate kinase

This enzyme requires Mg²⁺ to make its catalytic activity. The name derives from the reaction of the enzyme in the reverse direction, which occurs during photosynthetic CO₂ fixation. It is responsible for the production of the first molecules of ATP in glycolysis. Its amino acid sequence has to be extremely conserved in different organisms. The monomeric enzyme is composed of two domains of equivalent size, which corresponds to half N-and C-terminal. The substrate (1,3-bisphosphoglycerate) binds to the first half, while ADP binds to the second. Presents a sequential kinetic mechanism in which catalysis occurs by a proximity effect.
8^th enzyme 8th – Phosphoglycerate mutase

The phosphoglycerate mutase is dimeric, with each of its subunits with about 32kDa. As the name implies, this is a mutase enzyme, ie, catalyzes the transfer of phosphoryl groups within a molecule. In other words, it changes the position of phosphoryl groups. In fact, the enzyme is phosphorylated (fosfoenzima is one), and will give up its phosphoryl group to the carbon of the substrate 2, resulting in an intermediate with two phosphoryl groups (2,3-bisphosphoglycerate). Only after this step, is that the phosphoryl group that was originally in the substrate (position 3) is removed, regenerating the initial form (phosphorylated) enzyme.

The phosphoglycerate mutase has three different isoforms (isozymes or isoenzymes), predominantly found in cardiac muscle, skeletal muscle and the third one in the other tissues.

9th enzyme – Enolase

The enolase is a dimeric metalloenzyme, and each subunit has about 40-50 kDa. These subunits have an antiparallel orientation, interacting with each other via two salt bridges, involving an arginine and a glutamate each. The N-terminal domain of alpha-3 subunit has four helices and beta sheets. The C-terminal domain has two beta sheets and two alpha-helices, and it ends with a barrel consists of beta sheets and alpha helices alternate. The two Mg²⁺ ions required for catalytic activity are critical in neutralizing negative charges. This enzyme has a pH optimum of about 6.5, and can also be called fosfopiruvato dehydratase. It was initially discovered in 1934 by researchers Lohmann and Meyerhof. As with the enzyme before the enolase also has three different isoforms, of which one is predominantly found in muscle tissue, the other in neurons and the third one in the remaining parts of the body.
The enolase is inhibited by fluoride ion, and this fact is exploited, for example, when collecting blood samples for analysis. In this case, when it is important to inhibit glycolysis (to keep unchanged the concentration of serum glucose), blood can be collected in tubes containing fluoride.
10^th enzyme – Pyruvate kinase

This enzyme is responsible for the second ATP production in glycolysis and is the third regulatory enzyme of this pathway. It needs the presence of two metal ions: K⁺ and Mg²⁺ (or Mn²⁺). It has four different isoforms, one located predominantly in the liver, another in red blood cells, the other in cardiac and skeletal muscle and brain and the latter is mainly found in fetal tissues. It is a tetrameric enzyme, each subunit has about 500 amino acids.

Main bibliographic sources:

- Voet D, Voet JG, Biochemistry, Wiley

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers

Metabolic map on the utilization of different monosaccharides in glycolysis

Glycolysis serves to degrade different monosaccharides besides glucose. Here is a metabolic map that illustrates this situation.

http://www.biocarta.com/genes/Metabolism.asp

Glycolysis (enzymes of the preparatory phase)

As I have already mentioned on other posts, glycolysis is composed by 10 biochemical reactions catalyzed by enzymes all different. Today I dedicate this post to some information on the enzymes of the preparatory phase.
1^st enzyme - Hexokinase
This enzyme, present in all our cells, has different isoforms present in our body and is the first point of regulation of glycolysis. In general, regardless of the isoform considered, its mass is about 100kDa. It is an enzyme that can be structurally divided into two halves with plenty of homology, the N-terminal half and half C-terminal. Because of this characteristic, it is thought that the gene for this enzyme may have arisen by duplication of an ancestral gene. The 3D structure of hexokinase can be compared to the shell of a bivalve...

There are four major isoforms of hexokinase (I-IV), and the fourth may also be called glucocinase (or hexokinase D), and is found primarily in the liver. Glucocinase The kinetic properties and regulatory features significantly different from other isoforms. The hexokinase I-III have a very high affinity for glucose (Km for glucose is about 0.1 mM), and to a normal concentration of glucose (4-5 mM) the enzyme is saturated with substrate. That is, the amount of available substrate is sufficient for the enzyme to function at its maximum speed. On the other hand, glucocinase presents a much higher Km (10 mM), which means that under normal conditions the enzyme is far from saturated with substrate. Probably at this point you will ask: "What's the point of this? It should be much more advantageous to have an enzyme to function at its maximum speed!" The answer to this question is very simple ... The function of glucocinase is to produce glucose-6-P which is then diverted mainly to the synthesis of liver glycogen. Thus, it only makes sense we have a lot synthesize glycogen when glucose availability is high. Therefore, the glucocinase will only start operating at a higher speed if there is an increase in the substrate availability. In other words, unlike what happens with the other hexokinase, when higher the concentration of glucose increased the speed of action of glucocinase.

The main substrate of hexokinase is D-glucose, but can also use other substrates such as hexoses, such as D-fructose and D-mannose. However, the value of Km for these substrates is higher, ie, the enzyme can use them but has less affinity for the same. This situation occurs mainly caters for hexokinase I-III, and the glucocinase is more specific for glucose.

The mechanism of action of hexokinase is called the Random Bi Bi, in which the enzyme forms a ternary complex with glucose and the Mg2 +-ATP before the reaction occurs. It makes a catalysis by proximity effect.
2^nd enzyme - Fosfohexose isomerase

This enzyme has an activity highly dependent on pH, suggesting a mechanism of action involving charged side chains of amino acids in its active center. In fact, the presence of a glutamate and a lysine in the active site of fosfohexose isomerase is essential for the catalytic activity of the same. This enzyme is highly steroespecific.

3^rd enzyme - Phosphofructokinase-1 (PFK-1)

The PFK-1 is the second regulatory enzyme of glycolysis, and is its main point of regulation. Presents a certain analogy with hexokinase, because the reaction is identical to that catalyzes the first reaction of glycolysis. At the structural level, it presents as a homotetramer.

There is another PFK, the PFK-2, which does not act directly in glycolysis, but is central to its regulation, because it controls the levels of fructose-2 ,6-bisphosphate, an important activator of PFK-1! (I will soon put a post on the regulation of glycolysis ...)

4^th enzyme - Aldolase

This enzyme is highly steroespecific. Presents three different isoforms (A, B and C), whose expression varies during the development of the organism. The major isoform in humans is the isoform A.

n glycolysis, the aldolase catalyzes a reaction known as retro-aldol condensation. There are two amino acid residues essential for the activity of the enzyme, a lysine and a cysteine.

5^th enzyme – Triose phosphate isomerase

The triose phosphate isomerase appears as a homodimer. Each subunit has a barrel-shaped structure, composed of eight alpha helices and eight parallel beta sheets. It was the first enzyme discovered to exhibit this type of barrel alpha / beta. This enzyme has a high dependence on catalytic function of pH, which indicates that performs an acid-base catalysis. In fact, there are three amino acid residues essential for its activity, a glutamate, a histidine and lysine. These amino acid residues play a role towards the establishment of hydrogen bonds that stabilize the transition state. Additionally, the enzyme has a loop with 10 amino acid residues highly conserved. This loop is important to stabilize the enediol (intermediate reaction) formed during the catalytic activity of the enzyme.

Triose phosphate isomerase is often mentioned as a case of "catalytic perfection", since it has a reaction rate controlled by diffusion. That is, the product formation takes place in a way as fast as the collision of the enzyme and substrate which limits the speed and is even spreading the product out of the active site of the enzyme.

Main bibliographic sources:

- Voet D, Voet JG, Biochemistry, Wiley

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers