Enzymes

Enzymes are biological catalysts and therefore catalyze biochemical processes in living organisms. However, they can also function extracellularly – that is, outside the cell or organism. A catalyst increases the reaction rate of a chemical reaction without itself being transformed and without altering the chemical equilibrium. Enzymes act as catalysts by creating a physical and chemical environment that promotes the given reaction to proceed. They do this by binding to substrates, thereby increasing the local concentration of substrates, as well as having reactive functional groups that can participate in the reaction. Enzymes thereby lower the activation energy and increase the reaction rate of biochemical reactions. For this reason, enzymes are essential for all life on Earth. They catalyse reactions in the metabolism of cells that would otherwise be too slow – up to millions of years – if they were to take place without the presence of enzymes.

The two most important points when it comes to the structure and function of enzymes are:

1. Enzymes bind to their substrate with high affinity and specificity.
2. When the substrate binds to the active site, it causes structural changes in the enzyme.

When a substrate binds, there will be a change in the intermolecular bonds in the enzyme. These changes in structure promote the formation of the product of the reaction. Although some changes cause major changes in the entire enzyme, most occur in or around the active site.

The Different Main Classes of Enzymes

Most proteins whose function is to be enzymes have the ending -ase. In addition to the suffix, the substrate, or a description of the biochemical function the enzyme performs, is most often included in the name. For example, the hydrolase, peptidase, has its name as it breaks the peptide bonds in proteins by hydrolysis.

Enzymes are classified in a system according to 6 types of enzymatic reactions that enzymes catalyze, and they are therefore called as follows: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. For these 6 main classes, there are many subclasses for each of them. In this teaching material, we will focus on hydrolases, which are further divided into subclasses according to the type of bond they break, and then according to their type of substrate. Table 1 shows an overview of the main classes of enzymes.

Table 1:

The structure of proteins and the 20 amino acids

It is important to first understand the structure of proteins if you want to know how the structure of enzymes relates to their function and catalytic mechanism.

Proteins are polymers of amino acids. When we talk about amino acids in proteins, we are referring to the 20 common amino acids (Figure 12) that are coded for in DNA. In fact, there are over 140 amino acids, as these 20 amino acids can be modified after translation. But here the focus will only be on the 20.

As shown in Figure 11, all amino acids have a central carbon atom that is bonded to a hydrogen atom, a charged primary amine (NH₃⁺), also called an amino group, as well as a charged carboxylic acid group (COO-), and a variable seat chain R.

Figure 11: Structure of amino acid.

It is the variable side chain that characterizes the individual amino acid. The 20 amino acids can be divided into 4 different main groups according to the properties of their variable side chain. The overview in Figure 12 shows how the amino acids are divided into polar (hydrophilic), nonpolar (hydrophobic), and according to their electrical charge at a pH value of 7 and are therefore either basic (positively charged) or acidic (negatively charged). It is seen that glycine is the only amino acid that does not have a stereocenter, since its variable side chain is a hydrogen atom. Glycine is therefore also the simplest of all the amino acids.

Figure 12: Overview of the 20 amino acids divided by the chemical properties of their side chain. Download the overview as a PDF here.

Amino acids are bound to each other by a specific type of covalent bonds, called peptide bonds. The peptide bonds are formed by a condensation reaction. Here, a covalent bond is formed between the amino group in one amino acid and the carboxylic acid group in the other, and water is decomposed. When two or more amino acids bind together, it is called a peptide, and for many amino acids, it is called a polypeptide. When polypeptide chains are formed, there will always be a free amino group at the front end and a free carboxylic acid group at the back end of the polypeptide. The front end of a polypeptide is therefore called the N-terminal after the nitrogen atom in the free amino group, while the back end is called the C-terminal after the carbon atom in the free carboxylic acid group.

Figure 13: Condensation reaction where a peptide bond is formed.

Protein Structure Levels

The largest component of proteins is made up of polypeptide chains. This is called the primary structure of proteins. The primary structure describes the sequence of amino acids in the polypeptide chain from the N-terminal to the C-terminus. The sequence of amino acids determines the three-dimensional structure and biochemical function of the protein.

There are 3 different secondary structures; α-helixes, β-sheets, and β-loops. These are local structures in the protein, formed by bonds between peptides that are close to each other in the polypeptide chain. The main chain of the polypeptide (the part that is not variable) is polar, as the peptide bond contains a hydrogen donor, NH, and a carbonyl group (C=O), which is capable of forming hydrogen bonds. This polarity is a problem if a hydrophobic environment is to be created, and to overcome this problem, α helixes and β sheets are formed. Their formation neutralizes the polarity by forming hydrogen bonds between the NH and C=O groups in the peptide chains.

In α helixes, the carbonyl group forms a hydrogen bond with H from the peptide bond, which is located four amino acid units further down the chain. This forms a right-turning helix. At the ends of α helixes, there is a C=O and NH group that do not form bonds. These two groups are polar, and therefore you usually see the ends of helixes near the surface of the protein. These helixes are typically from 4 to over 40 peptides long in globular proteins.

In β-sheets, hydrogen bonds are also formed between the NH and C=O groups in the peptide bonds, but between chains that lie parallel to each other. β-sheets therefore form a flat structure, where the parallel chains are typically 5-10 amino acid units long.

α-helixes and β-sheets are connected by β-loops, which typically lie on the surface of the protein. This is because no hydrogen bonds form between NH and C=O in β loops. The side chains are freely available to form hydrogen bonds in the environment in which the protein is located.

Figure 14: The figure shows the three types of secondary structures. Hydrogen bonds (yellow dotted lines) are formed between oxygen from C=O (red) and NH groups (blue).

The tertiary structure of proteins is their overall three-dimensional structure, which is made up of bonds between the variable side chains of the amino acids. Proteins fold in the most energy-beneficial way. That is, it forms the structure that requires the least energy to maintain. The strongest type of bond in the tertiary structure is covalent bonds in the form of disulfide bonds between sulfur atoms in two cysteines. This is followed by ionic bonds, which are formed by amino acids with differently charged side groups. This could be between the negatively charged aspartate/aspartic acid (D) and the positively charged lysine (K). The second strongest bonds are hydrogen bonds. For example, hydrogen bonds can form between amino acids with an alcohol group in their side chain, such as serine and threonine. The weakest bonds are the hydrophobic interactions between the non-polar side chains of the amino acids

Figure 15: The figure shows the different types of bonds that can form between the amino acid side groups

The hydrophobic effects are a primary driver for the protein to acquire its spherical (spherical) structure. For water-soluble proteins, which are found in the cytoplasm, for example, they are seen to have a hydrophilic surface, as this is in contact with the surrounding environment and a hydrophobic interior.

Many proteins are made up of more than one polypeptide chain. The polypeptide chains can be either the same or completely different. With such proteins, the neighborhood structure is also considered. Here, the various polypeptide chains that are part of proteins are referred to as subunits. The quaternary structure is therefore the spatial distribution of subunits that are part of proteins.

Illustration of proteins

Proteins are often illustrated in different ways depending on what you are interested in studying about the protein. In Figure 16, the same protein is shown in 3 different ways. Sticks are mostly used if you only look at individual parts of the protein up close. The secondary structure is really important for how proteins fold, and therefore a very frequent way to illustrate proteins. The surface of the protein shows the part of the protein that is available to other molecules or the solvent in which the protein resides.

Figure 16: The structure of a peptidase from the bacterium Bacillus cereus shown in three different ways. The protein is colored depending on whether the individual amino acids are part of a α-helix (pink), a β-sheet (orange) or a β-loop (white).

The function of enzymes

The function of enzymes is to convert substrates, which can be either a single or several molecules, into one or more products. It was previously thought that the way enzyme and substrate were bound could be explained by a lock and key model. In this model, the enzyme and substrate fit together structurally instantly, like a lock and key. The part of the enzyme that binds to substrates and where the reaction takes place is called the active site (or active center). However, the lock and key model cannot explain how enzyme activity is regulated by the cell. Or how substrates can manage to bind to an active site that lies deep inside an enzyme.

Enzymes are dynamic and not static, as the amino acid side chains are standing and moving a little bit all the time. Later research has shown that enzyme and substrate bind in a slightly more complex way, explained by the induced-fit model (see Figure 17). In this model, both enzyme and substrate change structurally, allowing enzyme and substrate to bind. At the same time, this bond is not too strong, which ensures that the products can also be released again.

In the induced-fit model, an enzyme-catalyzed reaction proceeds as follows:

1. Enzyme (E) and substrate (S) are in the same environment.
2. The enzyme and substrate are initially bound and form an enzyme-substrate complex, ES.
3. The enzyme and substrate are changed by induced fit. Here, the chemical reaction really begins, and the substrate is in a transition state. This is also called the transition state intermediate. This is an unstable stage where the substrate is in the process of being chemically altered. The substrate is in a state between reactant and product. The universal symbol for the transition state is ‡. Here we have an activated complex EX, where the substrate is no longer in the same configuration as before the reaction started.
4. After being in the transition state, the substrate is converted into product(s) but still bound to the enzyme. An enzyme-product complex EP has been formed.
5. The products are released from the enzyme, and the enzyme is now freely able to react with a new substrate.

Figure 17: Induced fit model shown in 5 steps. Notice that the substrate changes intramolecularly, as it is in transition state EX^‡ in step 3.

The model in Figure 17 describes a simple enzyme-catalysed reaction in which a substrate is split into two products. It’s important to remember that some enzymes can also bind two substrates and convert them into one product. The fact that enzymes stabilize the substrate in the transition state plays a major role in lowering the activation energy for biochemical reactions. This is exactly why enzymes are so effective.

The active site

Although an enzyme and substrate adapt to each other when they bind, enzymes are still very specific. Enzymes therefore only catalyse one particular biochemical reaction. Their specificity is determined by its three-dimensional structure and its active site.

The active site orients the substrates so that they get the optimal orientation to enter into a reaction with the functional groups in the amino acids’ side chains.

The amino acids in the active site determine whether or not they can bind and enter into a reaction with a molecule. The amino acid side groups in the active site have functional groups whose chemical properties help to flip and orient the substrate so that the reaction can proceed. Figure 18 shows that two substrates fit into an active site, so that ionic bonds are formed between the charged side chains with the active site. The substrates also fit into the active site, so the hydrophobic parts come together.

Figure 18: The chemical environment of the active site is suitable for the substrate(s) to which it binds. The active site in the enzyme also makes sure to orient the substrates so that they can react with each other and form the product.

An active site is actually divided into two; a bonding part and a catalytic part. The bond part consists of a number of amino acid side groups, the purpose of which is to form the intermolecular bond with a substrate. The substrate is thus bound to the enzyme. During binding, the substrate is also rotated so that its configuration is optimal for its functional groups to interact with the active site. It is in the catalytic part that the reaction takes place. Here, bonds may be broken, and electrons and atoms are moved around to form one or more products.

Cofactors and coenzymes

Enzymes are not always made up of amino acids alone. Most often, enzymes require the presence of small molecules in the active site, called cofactors, in order for the catalytic reaction to take place. Cofactors are non-proteins that are directly involved in the catalytic mechanism. They either stabilize the enzyme, substrates, or help transform a molecule into a new one. They can be inorganic metal ions such as Fe²⁺, Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ or organic molecules such as vitamins. If the cofactors are organic molecules, they are called coenzymes instead. The role of the cofactor or coenzyme in the reaction is to transfer either electrons or atoms. If an enzyme has a bound cofactor, it is called a holoenzyme, if the cofactor is removed, the enzyme is called an apoenzyme.