Introduction to Enzyme Mechanism of Action
Enzymes are the protein substances that are needed for all the biochemical reactions taking place in the living cells. They catalyse chemical reactions that are essential for life. They are involved in processes like digestion and breakdown of nutrients, metabolism of nutrients to provide energy, synthesis of proteins, DNA, and other biomolecules, etc. They are also required for the synthesis of membranes, cells, and tissues. Enzymes are also involved in the processes of life such as motility, muscle contraction, and neural functions, etc.
During a chemical reaction, enzymes catalyse the conversion of one or more substrates into a product. They decrease the amount of energy required for a chemical reaction to take place. Enzymes are highly specific for their substrate. They also require highly optimum temperature and pH conditions for their proper action.
Different models have been proposed to explain the mechanism of enzyme action. In this article, we will study some properties of enzymes and try to understand the mechanism of action of enzymes through various models of catalysis. We will go through the classification of enzymes based on the type of reaction they catalyse. Different substances that help enzymes in their mode of action as well as factors affecting enzyme action will also be discussed.
Most of the enzymes are globular proteins made up of thousands of amino acids. The polypeptide side chains are folded onto themselves to form a compact globular structure. Each enzyme has a cleft or space where the catalytic process takes place, called active site or catalytic site.
The active site is a special cleft or space in an enzyme molecule formed by the folding of the polypeptide chain. It is a site where the substrate binds and the catalytic process takes place. It is lined by amino acids that participate in the process of catalysis.
As the substrate binds to the active site, an enzyme-substrate (ES) complex is formed. The formation of the ES complex induces some changes in the active site that starts the catalytic process. Soon, the process is completed, and an enzyme-product (EP) complex is formed. This complex subsequently disassociates, and products of the reaction are released.
Knowing the properties of enzymes provides an important basis to understand the mode by which enzymes act on different substances. Some important properties of enzymes are mentioned below.
Enzymes require an aqueous environment for catalysis to take place. They are soluble in aqueous media with their polypeptide chains folded in such a way that enzyme action can take place. Enzymes have polar amino acids on their surface that increase their solubility in aqueous media.
Enzymes are highly specific in nature. One enzyme catalyses only one reaction or a set of similar reactions. The active site of an enzyme recognizes and binds only one specific substrate molecule. Therefore, it can catalyse reactions specific to that substrate only.
Enzymes need specific temperatures for their maximum activity. This temperature is called optimum temperature. Any increase or decrease in temperature from the optimum level makes the enzymes inactive. The enzyme activity returns when the temperature reaches the optimum level, again. Too much increase in temperature may cause the denaturation of enzymes that cannot be reversed by the reversal of temperature.
Enzymes in the human body need an optimum temperature of 37.5 oC.
Enzymes are also highly specific for the pH of the working environment. Even a slight change in pH can cause denaturation of enzymes, leading to their irreversible inactivation. Any change in pH affects the ionization of amino acid side chains that causes gross changes in the shape of the enzyme.
Each enzyme has its own specific optimum pH. For example, pepsin in the stomach works in acidic pH at 1.5-1.6, trypsin in the small intestine works in basic pH at 7.8-8.7, pancreatic lipase at 8.0, etc.
Preservation of Enzyme
In the process of catalysis, enzymes are not used up. They remain the same at the end of a reaction. The catalytic process also does not cause any change in the structure of the enzyme. Thus, enzymes are only temporarily used by a catalytic process. They are required in small amounts and can be used again and again.
Mechanism of Action
The mechanism of enzyme action can be studied from two different perspectives. According to one perspective, enzymes provide alternate energy pathways for a reaction to take place. The other perspective describes the role of the active site in the process of catalysis. A brief detail of each of them is given below.
Alternate Energy Pathways
During all chemical reactions, a high energy intermediate is formed between the reactants and products. The energy needed for the synthesis of this intermediate is called free energy of activation. The state at which a high energy intermediate is formed is called the transition state.
Free energy of activation or transition state acts as a barrier between the reactants and the products. For a chemical reaction to take place, all the reactant molecules must possess enough energy to cross this high energy state.
In the absence of enzymes, only a small number of molecules have this much energy to overcome the transition state. The rate of reaction depends on the number of such molecules. If the free energy of activation is low, more reactant molecules will be able to overcome the transition state and the rate of reaction will be higher.
Enzymes catalyse a chemical reaction by providing an alternate reaction pathway with low energy of activation. In this way, more reactant molecules can overcome the energy barrier and are converted to products. Thus, the rate of reaction is increased.
Role of Active Site
Active site not only acts as a binding site for substrates. Rather, the complex molecular machinery plays important role in the conversion of substrate into products.
As already mentioned, a transition state is formed between the reactants and products during a chemical reaction. The bonds in the transition state are not that stable as seen in the case of reactants or products. Active site binds the transition state and helps to stabilize its structure. Thus, more reactants can be converted to products and the rate of reaction is increased.
The active site also facilitates different models of catalysis as described in the next heading.
Types of Catalysis
Catalysis is the process by which the rate of reaction is increased. Enzymes catalyse chemical reactions in living organisms by employing models of catalysis. Four general types of mechanisms of catalysis are described below.
Catalysis by Proximity
A chemical reaction can only take place if two or more reactants come close to each other. They should come close to a distance that a bond can be formed between them, called bond-forming distance. If the concentration of reactants is high, they are closer to each other meaning that the rate of reaction will also be higher.
When substrate molecules bind to the enzyme at its active site, a locally high concentration region is formed. The substrate molecules at the active site are in close proximity to each other. They are oriented in ideal positions for chemical reactions to take place. This increases the rate of reaction to a considerable extent.
The ionizable functional groups in the side chains of amino acids present at the active site can also contribute to the catalysis by acting either as an acid or a base. This type of catalysis is called acid-base catalysis.
Acid-Base catalysis is classified into two types: specific and general.
In specific acid-base catalysis, only protons or hydroxide ions are the acid or base participating in the reaction. The rate of such reactions depends only on the concentration of protons or hydroxide ions. The presence or absence of proton donors (acids) or proton acceptors (bases) at the active site does not affect the rate of reaction.
In general acid-base catalysis, the rate of reaction is influenced by all the acids or bases present at the active site.
Pepsin is an enzyme that exhibits acid-base catalysis. It belongs to the family of aspartic protease enzymes. The active site of the enzyme carried two aspartyl residues that act as acid-base catalysts.
In the first step, one aspartyl residue acts as a base and accepts a proton. This results in the formation of a transition state intermediate. In the next step, the other aspartyl residue acts as an acid and donates a proton to the transition state intermediate. This causes the breakdown of the transition state with the release of products.
Catalysis by Strain
It is seen in enzymes that catalyse lytic reactions. These are the reactions that involve the breaking of covalent bonds. In lytic reactions, substrates bind enzymes at the active site in a specific state that is not favourable for the bond to be cleaved. This specific conformation resembles the transition state intermediate.
In this specific conformation, a strain is put on the target bond. As a result, the target bond is stretched. The strain weakens the bond and makes it easy to be cleaved.
It should be kept in mind that substrate is not always strained. Rather, there is a new theory that suggests that enzyme is strained more easily as compared to substrate molecules. This theory is based on the Induced Fit Model of enzyme actions.
Induced Fit Model
This model of enzyme action was proposed by Daniel E. Koshland in 1958. According to the Induced Fit Model, when substrate molecules bind to the active site of the enzyme, they induce some structural changes in the active site so that it can effectively bind the substrate molecules. The substrate molecules do not fit exactly into the active site of the enzyme. Rather, the active sites of enzymes are flexible to some extent and undergo confrontational changes. In this way, active sites can accommodate the substrate molecules.
Induced Fit Model also suggests that during the binding process, the active sites can also distort substrate molecules, causing them to achieve a transition state immediately.
Lock and Key Model
It was proposed earlier by Emil Fischer in 1894. According to this model, the active site of an enzyme is a rigid structure and does not undergo any structural changes during the formation of the ES complex. Only those substrate molecules can bind to the active site that can completely fit into it.
This model was later rejected by Induced Fit Model.
During this process, a covalent bond is formed between the active site of the enzyme and substrate molecule. The enzyme participates in the catalytic process as a reactant.
The covalently modified state of the enzyme acts as a transition state. It provides an alternate pathway low energy pathway for the reaction to take place. Thus, the rate of reaction is tremendously increased.
At the end of the reaction, the enzyme returns to its original, non-modified state. Thus, the enzyme does not undergo any change in its structure and is again available for other similar reactions.
Cysteine, serine, and histidine amino acids at the active sites most commonly participate in this type of catalytic reaction.
Covalent catalysis is most commonly seen in group transfer reactions. It usually follows a ping-pong mechanism i.e. the product of the first substrate is released prior to the binding of the second substrate.
Chymotrypsin is a pancreatic protease released into the small intestine. It can be studied as an example of covalent catalysis. It belongs to the group of serine proteases.
It has a trio of serine, histidine, and aspartate at its active site. It forms a charge relay network or proton shuttle. The binding of the substrate at the active site starts the transfer of proton and an acyl-enzyme intermediate is formed. The peptide bond is cleaved, and the proton shuttle is reversed, restoring the enzyme to its original form.
Substances facilitating Enzyme Action
Many enzymes contain small molecules of metal ions that facilitate the action of enzymes in various ways. Such facilitating molecules are divided into three types as discussed below.
These are the molecular substances that are tightly bound to the enzyme. They are incorporated into the protein structure of enzymes either by covalent or non-covalent bonds.
Examples of prosthetic groups include pyridoxal phosphate, FAD, FMN, and biotin. Most common prosthetic groups constitute metal ions such as copper, iron, zinc, magnesium, etc. Enzymes containing metal ions are termed metalloenzymes.
These metal ions at the active site of enzymes can act as proton acceptors or donors facilitating acid-base catalysis. They also facilitate the orientation and binding of substrate to the active site and the formation of covalent bonds with the reaction intermediates.
These are the facilitating substances that reversibly attach to the enzyme or substrates at the active site. They do not become a part of the protein structure of enzymes.
Cofactors bind enzymes in a transient manner and can be easily disassociated. They perform functions similar to prosthetic groups. Cofactors must be present in the medium surrounding the enzyme for catalysis to take place.
Like prosthetic groups, most of the cofactors are also metal ions. Enzymes requiring metal ions as cofactors are called metal-activated enzymes.
These are the organic molecules that do not bind to enzymes. Rather, they act as a shuttle, transporting substances between the enzymes. They help enzyme action by transporting the substances needed for the stability or function of the enzyme.
Factors affecting Enzyme Action
The study of factors affecting the enzyme action also helps in understanding the mechanism by which enzymes catalyze chemical reactions. It also provides us with information about the conditions needed for optimum enzyme action. Some important factors are mentioned below.
Concentration of Enzymes
The rate of enzyme action varies directly with the concentration of enzymes, keeping the substrate concentration constant.
If the concentration of enzymes is decreased, the rate of reaction also decreases.
Increasing the concentration of enzymes increases the rate of reaction but soon a maximum limit is reached. It happens when all the substrate molecules are already bound to the enzymes and no additional substrate molecules are available to be converted into products. At this stage, the increase in enzyme concentration will not bring any change in the rate of reaction.
Concentration of Substrates
A direct relationship is seen between the concentration of substrate and the rate of reaction. Keeping the enzyme concentration constant, the rate of reaction increases or decreases with an increase or decrease in substrate concentration, respectively.
If the concentration of substrates is increased, the rate of reaction goes on increasing until a maximum limit is reached. After that, any increase in substrate concentration will not bring about any change in the rate of reaction. It is because, at this stage, all the active sites are occupied by substrate molecules. No free active sites are available for new molecules to be converted into the products.
Effect of Temperature
At low temperatures, the reaction takes place at a very low rate due to the limited movement of molecules.
As the temperature increases, molecules acquire kinetic energy and the collisions between them increase. As a result, the rate of reaction also increases. The maximum rate of reaction is achieved at a temperature called optimum temperature. The optimum temperature for enzymes in the human body is 37.5 oC.
If the temperature is increased above the optimum temperature, the kinetic energy increases to the extent that bonds among the molecules start breaking. The enzymes get denatured and cannot perform their action. So, the rate of reaction slows down and eventually, at a much higher temperature, the reaction stops.
Effect of pH
As mentioned earlier, enzymes work best at their optimum pH that is different for each enzyme. The rate of reaction is also maximum at the optimum pH. Even slight variation in the pH affects the ionization of amino acids at the active sites.
Change in ionization state changes the shape of the active site, the substrate molecules can no longer fit in, and the rate of reaction decreases or even stops.
Enzymes are organic molecules that catalyse the chemical reactions taking place in living organisms.
Enzymes have a cleft like space called the active site. The active site recognizes the substrate molecules, bind to them, and brings about their conversion into the products. The amino acids forming the active site participate in the process of catalysis.
Enzymes are soluble in polar solvents and work in aqueous environments.
Only one specific substrate or similar substrates can bind to the active site of an enzyme. There is a separate enzyme for each different substrate.
Enzymes need the optimum temperature to carry out the maximum catalytic process.
Optimum pH is also necessary for the action of enzymes.
Enzymes can be recycled and used again and again, as they are not consumed in the catalytic process.
A high energy transition state is formed during the conversion of substrates into products. Enzymes facilitate the formation of transition states by lowering the free energy of activation, by providing alternate energy pathways.
Active site stabilizes the transition state and plays an important role in enzyme action.
Substrate molecules are in close proximity to each other at the active site increasing their chances to react and form products.
The ionizing groups at the active site can act as proton donors or acceptors and thus can take part in acid-base catalysis. It can be specific or general. The action of pepsin in the stomach is an example of acid-base catalysis.
The active site can strain the substrate molecules causing them to form a transient state, called strain catalysis. The induced-fit model suggests that substrate and the active site can induce confrontational changes into one another that facilitate the catalytic process. In opposition to this, the lock and key model suggest that the active site and substrate have rigid shapes that cannot be changed.
Certain substances facilitate the enzyme action such as prosthetic groups, cofactors, and coenzymes.
The rate of enzyme action varies with changes in the concentration of enzymes, concentration of substrate, temperature, and pH.
Frequently Asked Questions
What are the 4 steps of enzyme action?
Enzymes first identify the substrate present in their vicinity and then bind it to form an enzyme-substrate complex. In the third step, an enzyme catalysed reaction takes place, and finally, the product and enzymes are released. Enzymes are not consumed or used up in this process.
What is an active site in enzymes?
The active site is a cleft or special pocket present in the enzyme where the substrate binds to form the enzyme-substrate complex. It is lined with amino acids that are suitable to make bonds with the substrate molecules.
What are the types of inhibitors of enzymes?
Enzyme inhibitors are of two types; competitive and non-competitive. Competitive inhibitors compete with the substrate molecules for the active site and inhibit enzymes by occupying their active sites. On the other hand, non-competitive inhibitors inhibit the enzyme action by binding to a site other than the active site.
How does a change in pH affect enzyme action?
Change in pH of the environment results in changing the structure of the active site of the enzymes. The enzyme structure is deranged and the enzyme action stops.
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