Mechanism of Action of Enzumes
Emil Fischer’s model Lock and Key model 1890.
Lock: Key model of enzyme action implies that the active site of the enzyme is complementary in shape to that of its substrate, i.e. the shape of the enzyme molecule and the substrate molecule should fit each other like a lock and Key
In 1958, Daniel Koshland, postulated another model; which implies that the shapes & the active sites of enzymes are complementary to that of the substrate only after the substrate is bound.
Figure: Models of enzyme- substrate interactions
Figure: Models of enzyme- substrate interactions
Mechanism of Enzyme Action (1913)
Michaels and Menten have proposed a hypothesis for enzyme action, which is most acceptable. According to their hypothesis, the enzyme molecule (E) first combines with a substrate molecule (S) to form an enzyme substrate (ES) complex which further dissociates to form product (P) and enzyme (E) back. Enzyme once dissociated from the complex is free to combine with another molecule of substrate and form product in a similar way.
ENZYMES ENHANCE THE RATE OF REACTION BY LOWERING FREE ENERGY OF ACTIVAION
A chemical reaction S P (where S is the substrate and P is the product or products) will take place when a certain number of S molecules at any given instant posses enough energy to attain an activated condition called the “transition state”, in which the probability of making or breaking a chemical bond to form the product is very high.
The transition state is the top of the energy barrier separating the reactants and products. The rate of a given reaction will vary directly as the number of reactant molecules in the transition state. The “energy of activation is the amount of energy required to bring all the molecules in 1 gram-mole of a substrate at a given temperate to the transition state
A rise in temperature, by increasing thermal motion and energy, causes an increase in the number of molecules on the transition state and thus accelerates a chemical reaction. Addition of an enzyme or any catalyst can also bring about such acceleration.
The enzyme combines transiently with the substrate to produce a transient state having c lower energy of activation than that of substrate alone. This results in acceleration of the reaction. Once the products are formed, the enzyme (or catalyst) is free or regenerated to combine with another molecule of the substrate and repeat the process.
Activation energy is defined as the energy required to convert all molecules in one mole of reacting substance from the ground state to the transition state.
Enzyme are said to reduce the magnitude of this activation energy.
* During the formation of an ES complex, the substrate attaches itself to the specific active sites on the enzyme molecule by Reversible interactions formed by Electrostatic bonds, Hydrogen bonds, Vanderwaals forces, Hydrophobic interactions.
Factors Affecting Enzyme Activity
Physical and chemical factors are affecting the enzyme activity. These include
- Temperature
- pH
- Substrate/enzyme concentration etc.
Temperature
Starting from low temperature as the temperature increases to certain degree the activity of the enzyme increases because the temperature increase the total energy of the chemical system .There is an optimal temperature at which the reaction is most rapid (maximum). Above this the reaction rate decreases sharply, mainly due to denaturation of the enzyme by heat.
The temperature at which an enzyme shows maximum activity is known as the optimum temperature for the enzyme. For most body enzymes the optimum temperature is around 370c, which is body temperature.
Figure. Effect of temperature on enzymatic reaction
1. Effect of pH
The concentration of H+ affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in an ionized or unionized sate in order to interact.For example, Catalytic activity may require that an amino-group of the enzyme be in the protonated form (-NH3+) At alkaline pH this group is deprotonated and the rate of reaction therefore declines.
Extreme pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid chains.
The pH at which maximum enzyme activity is achieved is different for different enzymes, and after reflects the pH+] at which the enzyme functions in the body.
For example, pepsin, a digestive enzyme in the stomach, has maximum action at pH 2, where as other enzymes, designed to work at neutral pH, are denatured by such an acidic environment.
Figure. Effect of pH on enzymatic reaction
Concentration of substrate
At fixed enzyme concentration pH and temperature the activity of enzymes is influenced by increase in substrate concentration.
An increase in the substrate concentration increases the enzyme activity till a maximum is reached. Further increase in substrate concentration does not increase rate of reaction.
This condition shows that as concentration of substrate is increased, the substrate molecule combine with all available enzyme molecules at their active site till not more active sites are available (The active Sites become saturated). At this state the enzyme is obtained it maximum rate (V max).
Figure. Effect of Concentration of substrate on enzyme activity
The characteristic shape of the substrate saturation curve for an enzyme can be expressed mathematically by the Michaelis Menten equation:
Vmax = Maximal velocity possible with excess of substrate
[S] = concentration of the substrate at velocity V
Km = michaelis-constant of the enzyme for particular substrate.
Relationship between [S] and Km
Km shows the relationship between the substrate concentration and the velocity of the enzyme catalyzed reaction.Take the point in which 50% of the active site of the enzyme will be saturated by substrate, Assume that at ½ Vmax-50% of the active site of enzyme becomes saturated. Therefore:
Vo = ½ Vmax, at 50% saturation
½ Vmax = Vmax[S]
Km + [S]
2[S] = Km + [S]
Km= [S]
Figure: Relationship between [S] and Km
Characteristics of Km
Km- can defined as the concentration of the substrate at which a given enzyme yields one-half its max. Velocity (i.e Km is numerically equal to the substrate concentration of which the reaction velocity equal to ½ Vmax)
Km- is characteristic of n enzyme and a particular substrate, and reflects the affinity of the enzyme for that substrate.
Km- values varies from enzyme to enzyme and used to characterized different enzymes.
Km- values of an enzyme helps to understand the nature and speed of the enzyme catalysis.
Small Km - A numerically small (Low) km reflects a high affinity of the enzyme for substrate because a low conc of substrate is needed to half saturate the enzyme- that is reach a velocity of ½ Vmax.
High Km - A numerically large (high) Km reflects a low affinity of enzyme for substrate b/c a high conc of substrate is needed to half saturate the enzyme.
High Km Value f an enzyme means the catalysis of that enzyme is slow compared to low Km.
Km does not vary with the concentration of enzyme.
Relationship of Velocity to Enzyme Concentration
The rate of the reaction is directly proportional to enzyme concentration at all substrate concentration. For example, if the enzyme concentration halved, the initial rate of the reaction (Vo) is reduced to one half that of the original.
Figure. Effect of Enzyme concentration on enzymatic reaction
Order of Reaction
When [S] is much less than Km, the velocity of the reaction is roughly proportional to the substrate concentration. The rate of reaction is then said to be first order configuration with respect to substrate. When [S] is much greater than Km, the velocity is constant and equal to V max. The rate of reaction is then independent of substrate concentration and said to be zero order with respect to substrate concentration.