ENOLASE: STRUCTURE, MECHANISM OF ACTION, FUNCTIONS - BIOLOGY - 2025

The enolase is the enzyme responsible for performing the conversion of D-2-phosphoglycerate (2PGA) phosphoenolpyruvate (PEP) in glycolysis and gluconeogenesis reverse reaction, two metabolic pathways are part of cellular energy metabolism.

The decision to catalyze this reaction in one direction or the other depends on the cell's access to glucose. That is, of the needs you have to adapt your metabolism to degradation or synthesis in order to obtain energy. Indispensable for the realization of their vital processes.

Three-dimensional structure of Enolase. By Jawahar Swaminathan and MSD staff at the European Bioinformatics Institute, from Wikimedia Commons.

Given that both metabolic pathways belong to the center of the central metabolic tree of living beings, it is not surprising that the amino acid sequence of this protein is conserved in archaea, bacteria and eukaryotes. And therefore that it has similar catalytic properties.

The localization of enolase in the cell is limited to the cytosol, a compartment in which both glycolysis (also called glycolysis) and gluconeogenesis take place in most organisms.

However, it has also been detected in other cell compartments such as the plasma membrane of many pathogens and cancer cells. There, it seems to be involved with the facilitation of cell dissemination processes, a function totally different from its classical function.

Enzymes capable of performing more than one function, such as enolase, are known as moonlighting enzymes.

Structure

The quaternary structure of enolase bound or not to its ligands has been determined in a large number of prokaryotic and eukaryotic individuals.

Each monomer has two domains: a small amino-terminal domain and a larger carboxyl-terminal domain. The N-terminal domain is made up of three α-helices and four β sheets. Whereas, the C-terminal is composed of eight β sheets that alternate between them forming a β-barrel that is surrounded by eight α helices.

Furthermore, two binding sites for divalent cations are found on each monomer which have been termed the "conformational site" and the "catalytic site." The first is not very selective and can bind a great variety of divalent cations in the absence of a substrate.

Whereas, the second binds to ions after the substrate has bound to the enzyme. The binding of ions to both sites is vital for the reaction to proceed.

Finally, it is important to mention that in homodimers, the monomers are joined maintaining a parallel orientation. Therefore, the active site is limited to the central region formed by said junction.

However, only residues of one of the two monomers participate in catalysis. This explains the ability of monomers to carry out the reaction under experimental conditions.

Mechanism of action

Mechanism of action used by the enzyme Enolase. By Kthompson08 at English Wikipedia, from Wikimedia Commons.

Structural studies, as well as those that have made it possible to determine the kinetic and physicochemical characteristics of enolase, have made it possible to understand its mechanism of action.

The way in which the enzyme catalyzes the reaction is quite interesting. Although only one substrate is involved, an ordered sequential mechanism is what has been proposed.

This begins with the binding of an Mg2 + ion to the conformational site of one of the monomers. It continues with the binding of the substrate to the active site followed by the binding of a second ion to the catalytic site and concludes with the prompt release of the product once the reaction has been carried out. At this point, the Mg2 + remains attached to the conformational site.

Along the same lines, to promote the reaction, the enzyme first mediates the generation of a carbanion intermediate, eliminating a proton from carbon 2 of 2PGA. It does this thanks to the action of a basic amino acid residue.

Sequentially, the removal of the hydroxyl of carbon 3 occurs by the action of an acid residue of the enzyme. At this point, the union of both carbons is carried out by means of a double bond forming PEP. In this way the reaction is terminated.

Features

Many of the enzymes studied so far are capable of performing a great variety of functions unrelated to their "classical function" in different cell compartments. These enzymes have been referred to as "moonlighting" enzymes.

In this sense, enolase can be considered as a moonlighting enzyme, since numerous functions opposed to its classical function have been attributed to it to date in both bacteria and eukaryotes.

Some of these functions are as follows:

- Participates in the maintenance of cell shape as well as in vesicular traffic by interacting with cytoskeletal proteins.

- In the nucleus of mammalian cells, it acts as a transcription factor that regulates the expression of genes associated with cell proliferation. It cooperates in maintaining the stability of mRNAs in the degradosome in bacteria.

- In pathogens, such as Streptococcus pneumoniae and Trypanosoma cruzi, it appears to act as an important virulence factor.

- It has also been found that in Streptococcus pyogenes, enolase is excreted to the extracellular environment, facilitating tissue degradation and evasion of the immune system in the host.

- It is expressed on the surface of tumor cells, enhancing metastasis.

Eolase and its relationship with the mechanisms of cell dissemination

Many pathogens, as well as tumor cells, express in their membrane or excrete proteases capable of degrading extracellular matrix proteins into the extracellular environment.

This ability allows these cells to break through tissues and spread rapidly throughout the host organism. Promoting in this way the evasion of the immune system and therefore, the establishment of the infection.

Although enolase lacks protease activity, it participates in the process of dissemination of many pathogens in its host as well as tumor cells during metastasis.

This is achieved thanks to the fact that it is expressed on the surface of these cells by functioning as a plasminogen receptor. The latter is the zymogen of a serine protease known as plasmin that is part of the fibrinolytic system and acts by degrading extracellular matrix proteins.

Therefore, surface-expressed enolase is a strategy that these cells have acquired to establish infection and spread successfully.

This strategy consists of two processes:

- Evasion of the host's immune system. Since these cells are coated with a host's own protein, they are ignored by the cells of the immune system that recognize non-own proteins associated with pathogens.

- Post-activation dissemination of plasminogen in plasmin. Whose participation in the degradation of extracellular matrix proteins, then facilitates the rapid and effective dissemination.

References

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