Functioning of Enzyme and Nature of Enzyme Action: What actually are enzymes, and what role do they play in our bodies? Enzymes are proteins that are synthesised by living organisms and are responsible for a variety of metabolic and biochemical events in the body. They’re biological catalysts that help the body’s reactions go faster. Let us learn more about them. Enzymes are the major enablers of life. They create the necessary circumstances for biological reactions to occur quickly. A “catalyst” is the term scientists use to describe a chemical entity that boosts the rate of a reaction. In this article, we will learn about the functioning of enzymes and the nature of enzymes actions.

Definition

Fig: Enzymes

A complex biological catalyst synthesised by the body in its cells to regulate the body’s different physiological functions is known as an enzyme. Exoenzymes are enzymes that work outside of living cells, such as those found in digestive fluids and tears’ lysozymes. Endozymes are enzymes that work inside living cells, such as Krebs cycle enzymes, glycolysis enzymes, and so on.

The “substrate” is the element on which an enzyme acts, and the Enzyme is often named after the substrate by adding the suffix “ase” to the substrate’s name. Proteases, for example, are a group of enzymes that act on proteins, lipases are a group of enzymes that act on lipids, and maltase is the name of an enzyme that acts on maltose.

Physical Nature of Enzymes

1. Enzymes behave like colloids or high-molecular-weight compounds in terms of their physical properties.
2. At temperatures below the boiling point of water, enzymes are destroyed or inactivated.
3. Most enzymes in liquid media are inactivated at 60 degrees Celsius.
4. Dried enzyme extracts may withstand temperatures ranging from 100 to 120 degrees Celsius and even higher. Enzymes are thus thermos-labile.
5. Every Enzyme has a certain temperature range for optimum action, which usually falls between 25 and 45 degrees Celsius. Enzymatic action is highest at 37 degrees Celsius, and enzymes become inactive when the temperature rises above 60 degrees Celsius.

Chemical Nature of Enzymes

With the exception of recently discovered RNA enzymes, all enzymes are proteinaceous in nature (Sumner, 1926). A nonprotein group may be present in some enzymes.

1. Enzymes are a three-dimensional structure made up of a linear chain of amino acids.

2. The amino acid sequence determines the structure, which in turn indicates the Enzyme’s catalytic activity.

3. When heated, the structure of the Enzyme denatures, resulting in a decrease in enzyme activity, which is usually linked to temperature.

4. Enzymes are larger than their substrates, and their size varies, ranging from sixty-two amino acid residues in fatty acid synthase to an average of two thousand five hundred residues. Only a small part of the structure is engaged in catalysis, and it is located near binding sites.

5. The active site of an enzyme is made up of the catalytic and binding sites.

6. A limited number of ribozymes exist that act as biological catalysts based on RNA. It interacts with proteins in a complicated way.

The enzymes can be classified into the following groups based on their chemical nature:

(i) Simple Enzymes: Some enzymes are simple proteins, meaning they release amino acids when hydrolysed. Pepsin, trypsin, and chymotrypsin are examples of this type of Enzyme.

(ii) Conjugate Enzymes: Conjugated enzymes are the enzymes that comprise of a conjugated group attached to them.

Fig: Classification of Conjugate Enzymes

1. Enzyme with a protein part called apoenzyme (for example, flavoprotein) and a nonprotein part called the cofactor.
2. A holoenzyme is a full conjugate enzyme that consists of an apoenzyme and a cofactor.
3. Only when both components (apoenzyme and cofactor) are present can there be enzymatic action.
4. A cofactor is a nonprotein organic compound that is occasionally a simple divalent metallic ion (e.g., Ca, Mg, Zn, Co, etc.). Some enzymes, however, require both types of cofactors. A prosthetic group is formed when the cofactor is tightly attached to the apoenzyme.
5. Simple metal ions are one type of cofactor, whereas complex organic groups, often known as coenzymes, are another.
6. Prosthetic groups are cofactors that are tightly covalently or noncovalently linked with the protein.
7. Cytochromes, for example, are enzymes with porphyrins as their prosthetic groups.

(iii) Isoenzymes (Isozymes)

1. Isoenzymes are distinct isoforms of the same enzyme that catalyse the same process but have different physical or kinetic characteristics, such as isoelectric point, pH optimum, substrate affinity, or inhibitor effect.
2. Separate isoenzyme forms of the same Enzyme are frequently produced from different genes and can be found in various bodily tissues.
3. Lactate dehydrogenase (LDH), which catalyses the reversible conversion of pyruvate to lactate in the presence of the cofactor NADH, is an example of an enzyme with distinct isoenzyme forms. LDH is a tetramer made up of two types of subunits, H and M, that differ slightly in amino acid sequence.
4. The two subunits can randomly join to generate five isoenzymes with the following compositions: H4, H3M, H2M2, HM3, and M4. Electrophoretically, the five isoenzymes can be separated.
5. Isoenzymes have been discovered in over 100 enzymes. Thus, wheat endosperm alpha-amylase has 16 isozymes, human lactic dehydrogenase has 5 isozymes, and maise alcohol dehydrogenase has 4 isozymes.

(iv) Metallo-enzymes: Monovalent (K+) and divalent cations (Mg++, Mn++, Cu++) metal cofactors are used in enzymatic processes. These may be retained loosely by the enzyme or, in some situations, incorporated into the molecule’s structure. Metalloenzymes are enzymes that contain metal as part of the molecule, such as iron in haemoglobin or cytochrome.

Significance of Enzymes Action

In our bodies, enzymes serve a variety of purposes. These are some of them:

1. In diagnostic applications, the level of enzymes in serum is measured.
2. When specific enzymes are detected in the blood, it means that tissue or cellular damage has occurred, resulting in the release of intracellular components into the bloodstream.
3. As a result, when a doctor says they are going to test liver enzymes, the goal is to see if there is a risk of liver cell damage.
4. The aminotransferase alanine transaminase, ALT (also known as serum glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST (also known as serum glutamate oxaloacetate aminotransferase, SGOT) are commonly measured enzymes; lactate dehydrogenase, LDH creatine kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT.
5. Acute pancreatitis is an inflammatory condition in which the auto digesting of the pancreas is observed due to the activation of certain pancreatic enzymes.
6. To eliminate non-nutritive substances from the body, enzymes undertake a variety of biochemical reactions such as oxidation, reduction, hydrolysis, and others.

Mechanism of Enzyme Action

The enzyme promotes the progression of a reaction, but it remains unchanged after the completion of the process. Michaelis and Menten postulated in 1913 that during enzymatic activity, intermediate enzyme-substrate complex forms. The following scheme may be written to explain  the concept:

1. Enzymes are biological catalysts that change the kinetic characteristics of a reaction to speed it up. Thus, the Enzyme (E) catalyses the reaction between the substrate (S) and the enzyme-substrate complex (ES), where K1 is the rate constant for the formation of ES and K2 is the rate constant for the dissociation of ES into E and S.
2. Substrate (S) is transformed into products after the synthesis of ES, making an enzyme (E) available for subsequent combination with more substrate. The constant K3 can be used to represent the rate of conversion of ES to the reaction products.
3. Each enzyme-catalysed reaction has a distinct Km value, which is the Michaelis-Menten constant, which is a measure of the enzyme and substrate’s tendency to combine.
4. In this sense, the Km value is a measure of an enzyme’s affinity for a specific substrate. The lower the Km value, the higher the enzyme’s affinity for its substrate.
5. Enzymes have no effect on the equilibrium ratio of reactants and products since they do not initiate the reaction. Enzymes, on the other hand, accelerate the rate of reaction 108 to 1012 times in both directions to reach equilibrium.

Activation Energy

1. The kinetic or collision hypothesis asserts that in order for molecules to react, they must collide and have enough energy to surpass the reaction’s energy barrier.
2. The activation energy or energy of activation (G) is the lowest amount of energy required to overcome the energy barrier and change substrates into the transitional state.
3. The transition state is an unstable complex that forms at some point during the reaction between the substrate(s) and the products (P) and possesses the reaction pathway’s maximum energy. As a result, activation energy raises the kinetic energy of substrates, causing rapid collisions between Enzyme (E) and substrates (S). The G in a non-enzymatic reaction is extremely high, and no biological system can provide it.
4. For acid hydrolysis of sucrose, for example,  ∆G≠  is 32 Kcal/mole. Enzymatic hydrolysis of sucrose, on the other hand, requires just 9.4 kcal/mole of energy.
5. As a result, it is obvious that enzyme reduces the energy of activation (G) for a biological reaction by more than 50% for the reasons listed below:
   a. Increasing the substrate’s  ∆G,
   b. Maintains the transition state’s stability
   c. When substrates form bonds with the catalytic site, binding energy (kinetic energy) is released;
   d. The substrate’s bonds are weakened by a nucleophilic and electrophilic attack by the catalytic site.

Energy Change

Gibbs energy is the difference in energy levels between the substrate and the product (G). The  ∆G determines whether or not a reaction is thermodynamically favourable. The reaction has a negative ∆G value, indicating that it is energetically favourable. If the ∆G value is positive, the reaction is endergonic and not energetically favourable (require the input of energy). The rate of a reaction is unaffected by the ∆G of the reaction, but the rate of the reaction is regulated by the ∆G≠ . As a result, ∆G is distinct from ∆G≠ .

Theories of Enzyme Action

1.  Lock and Key Hypothesis

Emil Fischer proposed the enzyme-substrate complex in 1884, assuming a solid lock-and-key relationship between the two. The active site is the part of the enzyme where the substrate (or substrates) mix as it undergoes conversion to a product.

Reversibility of the reaction would not occur if the active site was rigid and particular for a given substrate because the structure of the product is different from that of the substrate and would not fit well.

E + S ⇋ ES→ E + P
(Intermediate Complex)
Where E = enzymes, S = substrate, P = product)

Reactions are reversible, as shown by the equation above. Only the rate of a process is influenced by enzymes, not the direction. The reaction will continue in either direction until it reaches equilibrium.

Fig: Lock and Key Hypothesis

This hypothesis assumes that a certain lock (substrate) can be opened using a specific key (Enzyme) that has been carefully designed to do so. Because the reaction or unlocking of the lock occurs here, the notched area of the key is equivalent to the active site. Enzyme specificity is accounted for by the lock and key. Only certain molecules can fit into specific active sites appropriately. As long as the engineering designs are identical, a key (enzymes) can open a variety of substrates (certain peptide linkages). As a result, when the complex is formed, the substrate fits perfectly into the enzyme’s active site, much like a key fits into a lock.

2. Induced-Fit Theory

In contrast to Fischer’s rigidly structured active site, Daniel E. Koshland (1973) discovered evidence suggesting a close approach of the substrate (or product) might cause the active site of enzymes to undergo a conformational change that facilitates a better combination of the two. The induced-fit hypothesis is a popular term for this concept, which is demonstrated below. Many occurrences of induced fit appear to change the structure of the substrate, allowing for a more functional enzyme-substrate combination.

Fig: Demonstration of Induced fit model

Factors Affecting Enzyme Action and Enzyme Kinetics

1.  Enzyme Concentration

The rate of a biological reaction increases as the enzyme concentration increases, up to a point known as the limiting or saturation point. An increase in enzyme concentration has little effect beyond that.

2. Substrate concentration

A. When the enzyme concentration is fixed, increasing the substrate causes a quick increase in the velocity or reaction rate.

B. However, when the substrate concentration rises, the rate of reaction slows until there is no further change in velocity at a high substrate concentration.

C. The substrate concentration required to give half the maximum velocity (Vm/2) may be easily calculated using the above diagram, and it is a crucial constant in enzyme kinetics.

D. It defines the Michaelis constant or Km. In other words, K is defined as the substrate concentration when V= ½ Vm-Under carefully defined conditions of temperature, pH, and ionic strength of the buffer, this constant Km approximates the dissociation constant of an enzyme-substrate complex. The reciprocal of Km or 1/Km approximates the affinity of an enzyme for its substrate.

3.  Temperature

An enzyme can only function within a narrow temperature range. They demonstrate enhanced reactivity with temperature as catalysts, but their proteinaceous nature puts them at risk of thermal denaturation above the optimal temperature.

4. pH

The pH at which an enzyme’s highest activity occurs varies a lot from one Enzyme to the next. This is known as the pH optimum. Any small change in either direction tends to reduce enzyme activity significantly. Because enzymes are proteins, pH changes affect the ionic character of the amino and carboxylic acid groups on the protein surface, which has a significant impact on an enzyme’s catalytic nature.

5. Hydration

Because the continuous phase is higher, the Enzyme acts best when the substrate’s kinetic activity is increased. That’s why seeds with little water content have limited enzymatic activity, despite the fact that they have plenty of substrates. However, at germination, the enzymatic activity increases dramatically, owing to the absorption of water and subsequent promotion of the kinetic activity of substrate molecules.

Kinetics of Enzyme Action

The Michaelis constant K is significant because it indicates the mechanism of action of an enzyme catalysing a process. It’s worth noting that for low substrate concentrations, the velocity–substrate relationship is nearly linear and follows first-order kinetics, i.e., the rate of the reaction A–>B is proportional to the substrate concentration [A].

V = K’ [A] low [substrate]

1. Where V is the observed velocity of the reaction at concentration [A]
2. K’ is the specific rate constant.

At high substrate concentration, however, the velocity of the reaction is maximum and is independent of the substrate [A]; hence it obeys the zero-order kinetics.

Vm = K’ Saturating [Substrate]

The Michaelis-Menten equation, which describes this relationship and also satisfactorily explains curve, is as follows:

V = Vm[S]/Km +[S]

1. Where V = initial reaction velocity at given substrate concentration [S]
2. Km = Michaelis constant, moles/litres
3. Vm = Maximum velocity at saturated substrate concentrations
4. [S] = Substrate concentration in moles / litre

Determination of Km of an enzyme reaction by Michaelis-Menten equation is in practice difficult. An outcome of this equation called the Line-weaver-Burk plot is often used for such determination.

Turn over Number

When the Enzyme is the rate-limiting factor, the turnover number is defined as the number of substrate molecules converted every minute by a single enzyme molecule.

Fig: Lineweaver burk plot

Summary

An enzyme is a complex biological catalyst produced by the body in its cells to regulate the body’s various physiological activities. It behaves like colloids or high-molecular-weight compounds in terms of their physical properties. At temperatures below the boiling point of water, enzymes are destroyed or inactivated. The enzymes can be classified into the following groups based on their chemical nature as Simple Enzymesare simple proteins, meaning they simply release amino acids when hydrolysed.

Enzyme with a protein part called apoenzyme (for example, flavoprotein) and a nonprotein part called the cofactor. A holoenzyme is a full conjugate enzyme that consists of an apoenzyme and a cofactor. Metalloenzymes are enzymes that contain metal as part of the molecule, such as iron in haemoglobin or cytochrome. Isoenzymes (Isozymes) are numerous molecular forms of an enzyme that occur in the same organism and have identical substrate action. In our bodies, enzymes serve a variety of purposes. Theories of enzyme action include the lock and key hypothesis and induced-fit theory. Factors affecting enzyme action and enzyme kinetics are enzyme concentration, substrate concentration, temperature, pH and hydration.

Frequently Asked Questions (FAQs)

Q.1. What is the function of enzymes?
Ans: Enzymes are proteins that assist our bodies to speed up chemical reactions. Enzymes catalyse a variety of chemical reactions involved in growth, blood coagulation, healing, illnesses, respiration, digestion, reproduction, and a variety of other biological functions.

Q.2. What are enzymes, and give some examples?
Ans: Proteins that serve as catalysts are known as enzymes. Enzymes are used by nature to speed up the transformation of one material into another—examples: Lipases, Amylase, Maltase and Trypsin.

Q.3. What are the steps involved in enzymatic action?
Ans: An enzyme attracts substrates to its active site, catalyses the chemical reaction by which products are formed, and then allows the products to dissociate (separate from the enzyme surface). The combination formed by an enzyme and its substrates is called the enzyme-substrate complex.

Q.4: What are 4 factors that can control or regulate enzyme activity?
Ans: Several factors affect the rate at which enzymatic reactions proceed – temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

Q.5: Is enzyme-specific in nature?
Ans: Enzymes are extremely specific in nature, meaning that only one Enzyme can catalyse a single process. Enzyme sucrase, for example, can exclusively catalyse sucrose hydrolysis.

Study About Structure Of Enzymes Here

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