Mechanism of enzyme action

Some amount of energy must be put in to get the reaction initiated. This energy is required to activate the substances to react. Hence, this energy is called activation energy. In the initial step of reaction, enzymes by functioning as catalysts, serve to reduce the activation energy required for a chemical reaction to take place. Without altering the process, enzymes speed up the overall rate of reaction.

Lock and key hypothesis:

Enzymes are very specific to particular substrate, it was suggested by Fischer in 1890. Specificity of enzyme on substrate because of enzymes have particular shape, into which substrate fit exactly. the substrate is imagined being like a key whose shape is complementary to the enzyme shape or lock. The site where the substrate binds in the enzyme is known as the active site which has the specific shape.

The enzyme catalytic process has been described in the following stages.

  1. Initially, the substrate binds to the active site of the enzyme, fitting into active state
  2. Binding of the substrate induces the enzyme to alter enzyme shape and it leads to fitting more tightly around the substrate.
  3. The active site of the enzyme, now in close proximity of the substrate breaks the chemical bonds of the substrate and the new enzyme-product complex is formed
  4. After the reaction, enzyme releases the final products by decreasing the affinity between enzyme and product.
  5. Finally, the enzyme is ready to involve in another enzymatic reaction again, this means enzymes can recycles the process by binding the other substrate molecule.

Mostly molecular structures of the enzymes are far larger than reactants (substrate) they act on. However, the active site in the enzyme structure is very small portion having between 3 to 12 amino acids. The remaining portion of the enzymes have around 20 to 200 and more amino acids, which make up the enzyme bulky and maintain the functioning portion (shape) of the enzymes correctly. This extra part of the enzyme is also very important if the active site is to function at the maximum rate. Once the enzyme converts the substrate to product, the product no longer fit into the active site, immediately releases into the surrounding medium. Leaving the active site free, welcomes the other substrate molecule to fit into it and which makes the other enzymatic reaction.

Components of the enzyme active site

The dynamic changes and motions in enzyme active site is very crucial during catalytic process. The chemical composition of the active site is important to access the substrate binding and controlling of solvent access during the reaction. Active sites of the enzymes have some sort of charges either it be negative or positive, termed as nucleophilic character. The nucleophilic character of the active site with any charge is developed by side chains of the amino acids present in the active site. Hydrophobic side chains of amino acids such as Ala, Val, Leu, Ile are present less frequently in active site pockets than in the entire protein. While, aromatic side chains of amino acids, Trp, Tyr, Phe, small polar side chains (Ser, Thr), and particularly Glycine, are found frequently in active site. The charges of the active site allow the binding of specific substrate to react (oxidation or reduction). Allowing the substrate and any solvent into the active site strictly controlled by gates, which are present in the active site or in front of catalytic cavity. The gates present in the active site contain amino acid side chains that can close or open an access pathway by rotating. For example, in some cases exclusion of water from some parts of the cavity, such as the active site or a specific tunnel of the enzyme, is necessary for functioning of numerous enzymes. In most of the enzymes, gates prevent the entering of solvent and protonating the ammonia present in the active site, which is very crucial to substrate binding access. In the enzyme cytochromes P450, the gate (water channel) controls hydration of the substrate in the active site, which is very important for cytochrome activity. Interestingly, in some enzymes, the gates may permit the solvent to enter only to a specific part of the cavity, eg. carbamoyl phosphate synthetase. In hydroxysteroid dehydrogenase, the gates may prevent the access of water molecules into the cavity when a substrate or a cofactor is not present. Gates also play a major role in filtering the potential substrate from non-specific substrate, thus they play an important role in controlling enzyme selectivity.

Some examples of enzymes which are having gates at the entrance to their active sites, including imidazole glycerol phosphate synthase, toluene-o-xylene monooxygenase, monooxygenase, acetylcholinesterase, type III polyketide synthases, choline oxidase, NiFe hydrogenases, carbonic anhydrases, formiminotransferase-cyclodeaminase, glutamate synthase, and FabZ β-hydroxyacyl–acyl carrier protein dehydratase.

Factors affecting the enzyme activity

Enzymes are mostly proteins (except some RNA based enzymes) with tertiary structures. Any alterations in the enzyme structure would affect the overall activity of the enzymatic reaction. Some of the abiotic factors can directly affect the enzymatic reaction.

(i) Hydrogen ion concentration on enzymatic reaction

Concentration of hydrogen ions (pH) in enzymatic reaction play a crucial role. Enzyme at certain pH shows a maximum activity, this pH is known as optimum pH of enzyme. increasing or decreasing of pH declines the overall activity of enzyme. Some enzymes function well in an acidic medium, other in alkaline medium.

 (ii) Temperature on enzymatic reaction

  1. Enzymes typically active (function) at certain range of temperature, however this range may be various from enzyme to enzyme depends on their origin.

Example:- Taq polymerase is heat stable DNA polymerase synthesized by thermophilic bacterium Thermus aquaticus. In research labs the name of the enzyme termed as Taq Pol or simply Taq. This enzyme can replicate DNA even at above 90°C and half-life of Taq is more than 2hours at 92.5°C.

  1. At particular temperature enzymes show maximum rate of enzymatic reaction is known as its optimum temperature. Which means enzymatic reaction decline at both below and above optimum temperature.
  2. Low temperature state enzymes are temporarily inactive and when the temperature raises to normal state then enzymes will gain their lost activity. This low temperature state generally used in laboratory conditions to preserve the enzymes for longer period. Higher temperature is enemy for most of the enzymes, at this state enzymes are denatured and losses their activity by destroying the week hydrogen bonds in their tertiary structure. These structural changes loss the enzyme catalytic activity. Once the enzyme (protein) is denatured it remains inactive even if the temperature is come back to normal state.

(iii) Substrate concentration on enzymatic reaction

The concentration of substrate in enzymatic reaction decides the overall rate of reaction. Increase in substrate concentration, increases the velocity of the reaction. In certain stage ultimately, rate of reaction reaches a maximum velocity (Vmax) at this stage there is no exceeded rate of reaction even if further raises in substrate concentration. This is because, in this condition all the enzyme molecules completely saturated with substrate. which means no active site is available for additional substrate molecules.

Michaelis constant

Michaelis constant (Km) is a mathematical derivation or constant which describes the substrate concentration at which the chemical reaction catalyzed by an enzyme attains half of enzyme maximum velocity or the concentration of the substrate at which half the maximum velocity of the enzyme reaction is attained.

 

 

 

 

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