Living cells need to perform a series of specific and strictly regulated chemical reactions to survive in the environment. The study of the intricate web of reactions that gives to the cell "life" is called metabolomic, but for today I want to focus on what is actually allowing cells to perform chemical transformations at very mild conditions of pH, temperature, and salt concentrations.

Enzymes are chemical catalysts, proteins that can take one or two starting compounds, usually referred to as substrate (S), to produce a particular product (P). Enzymes have a very complex structure and their activity is directly linked to the 3D conformation, which must be in its native form to preserve the biological function.


3D structure of alcohol dehydrogenase (Scaptodrosophila lebanonensis), on top the representation of local secondary structures, in the bottom animation, are highlighted the two chains that compose the enzyme.[4]

How enzymes really work

To be fair, even if enzymes sometimes seem sentients, they cannot do whatever they want they cannot "decide" the direction of the reaction, which is guided only by the equilibrium constant, they only act on the kinetic, also known as the speed at which the product is formed (or the substrate disappear). Each reaction is governed by the free energy levels of both substrate and product and the energy of activation. Higher is the “energetic jump” necessary to reach the substrate, slower is the reaction: imagine that the substrate have random energy fluctuations, higher jumps are less probable than lower ones, if the fluctuation is sufficiently high the substrate can reach the product state. Enzymes, by binding the substrate and arrange correctly its chemical components, can lower the energy of activation, accelerating the reaction.


Enzymes usually work only on a specific functional group on the substrate, let’s say a hydroxyl group (Chemoselectivity), but is able to distinguish the “right one” between the several that may be present in the macromolecule (Regioselectivity), like the -OH present in the 3rd carbon of glucose. This level of accuracy is necessary to be useful inside the cell, where hundreds of thousands of molecules are present at the same time, and is often a problem when we want to create very complicated molecules, through chemical synthesis. Another important aspect that enzymes usually take into account while performing their activity is the Stereoselectivity, that, in other words, is the ability to distinguish among different isomers of a molecule. The presence of different isomeric forms for a specific compound depends on different factors, like the presence of symmetries and the arrangement of the substituents in the space. The study of the different isomers is pretty complicated and require an entire post by itself, but is fascinating in particular when applied to biological systems, we will see an example of this later.[1]

The capability of the enzyme to selectively perform chemical reactions is given by its tridimensional structure, that in vivo is guided during the process of translation in the cell (synthesis of the proteins in ribosomes); in the majority of the cases, in the structure of the enzyme we can distinguish 2 domains:

1.     Active site: this is where the reaction takes place, and even if the enzyme or the enzyme complex can rich thousands of amino acids in length, the reaction is carried out usually by a few specific and spatially-oriented amino acid residues. This fold is often placed in the interior part of the protein, protected by the action of solvent.

2.     Recognition site: this is the region that selectively allows the entrance of the substrate in the active site, and the selection an be achieved by putting some barriers at the entrance, or by changing the chemical affinity that this region has towards the substrate (charges, hydrophobicity, polarity).

To be precise, the enzyme is not present in solution as an empty box, ready to be filled exactly by the right substrate, (a lock-key mechanism), but the presence of the substrate itself will induce a conformational modification in the enzyme that will provide the energy to take the substrate and perform the reaction.

But how exactly the reaction occurs? We can take a look at a simple example: beta galactosidase, a class of enzymes that cleave the glycosidic bonds present between two sugars (like the one present between glucose and galactose to form lactose). In this case, 2 amino acid residues are involved in the reaction, aspartate and glutamate both of which contain a carboxylic group. The reaction that takes place requires a molecule of water to break the bond between the two sugar, and is made by 4 main steps:

1.     Initially, the lactose is recognized and brought to the active site where aspartate is in its deprotonate state. The reaction takes place at neutral pH.

2.     Due to its positioning, glutamate is able to donate a hydrogen atom to break the glycosidic bond; at this point the glucose is free but the galactose is still attached to the enzyme

3.     Glutamate is able to take its hydrogen atom back, but this time from a water molecule which acts as the solvent in this reaction, in doing so glutamate goes back to its initial state, and the new specie OH- will attack the galactose.

4.     What originally was water now is part of the galactose molecule: its new hydroxy group in C1 position. The products can then be released and the process can start again


The mechanism is not too different from the one that can be performed chemically in acid conditions (in vitro), and at the end of the reaction we have exactly the same products, so, starting from 1 molecule of lactose we obtained 1 of glucose, 1 of galactose and we consumed 1 molecule of water (this is why it is called hydro-lysis). In some cases, the Asp residue can be substituted by another residue of Glu.[2]

Enzymes that can work for us

The huge collection of enzymes that cells of different organisms have, gives us a picture of the complexity that can arise from the process of evolution through natural selection over millions of years. What I’m really interested in now is, obviously, how to exploit what nature had created for more practical uses (selfish humans). The application of enzymes for Biocatalysis, even at industrial scales, is nothing new, the technology has been developed in the last 30 years, now the aim is to lower the costs, find new applications, increase the yields and find new sophisticated uses. Enzymes are responsible for a series of products that probably, you consume or use every day: syrups, food additives, pharmaceutical compounds, detergents, packaging, bioplastics, etc…

I want to report an example that summarizes how enzymes work and why they are important for us. Aspartame is a dipeptide, formed by aspartate and phenylalanine methyl ester, is commonly used as a low calories sweetener. At industrial level, it is synthesized starting from these two amino acids (that could be produced by fermentation), and using thermolysin, a protease enzyme that normally cleaves the bond between two amino acids. So, an enzyme that usually breaks a bond, is used to form a new one by fine-tuning the condition of the reaction.

To allow this we have to shift the equilibrium of the reaction towards the products, and this can be done by depleting the reaction mixture of one or more products at a time. That’s why large excess of phenylalanine is used: it is able to react with the synthesized aspartame forming a complex that precipitates out of solution (it also helps the recovery). Another product that we can eliminate is water (one of the most forgotten products in any type of reaction) by using salts or solvents.

What is even cooler in this reaction is that the enzyme is stereoselective towards L-phenylalanine (one of the two enantiomers of the molecule), so we can start with a mixture of unpurified amino acids (racemic mixture), and let the reaction going. Once the reaction is completed the “wrong” enantiomer can be recovered and a new mixture of 50/50 enantiomers can be easily resynthesized  (racemization) and used again. This can be useful if the phenylalanine is chemically synthesized, a process through which obtaining pure enantiomers is often difficult. What is also difficult to achieve chemically is to form the new bond between the amino group of phenyl alanine, and the right carboxylic group of aspartate: the reaction is regioselective in order to form the peptide with the alpha carboxylic group.[3]



 So, in the end, what is important to know about these special proteins? They speed up reactions that at physiological conditions won’t occur, they are also very specific, and help us in synthesizing large biomolecules, often making the processes simpler, cheaper and more environmentally friendly (which is always nice).

There’s so much to know about enzymes, like how to modify them, their stability in different environments, immobilization technics, inhibition, kinetic and so much more, that iI would like to explore in next posts.

Hope you enjoyed my first post here! I just want to bring attention to this subject that is often underrated and that I find fascinating.


  1. More definitions on selectivity (general biochemistry): https://www.quora.com/In-Organic-Chemistry-what-is-the-difference-between-Regioselectivity-and-Stereoselectivity
  2. The Wikipedia page of lactase: https://en.wikipedia.org/wiki/Lactase
  3. More informations about aspartame synthesis: Gabriel Birrane, Balaji Bhyravbhatla and Manuel A. Naviacorresponding, 2014. “Synthesis of Aspartame by Thermolysin: An X-ray Structural Study”, PMID: 24944748
  4. Take a look to the Alcohol dehydrogenase 3D structure on RCSB PDB: https://www.rcsb.org/structure/1B16
  5. Make your own PDB animation on https://proteopedia.org/wiki/fgij/