Factors Affecting Enzyme Activity



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 Factors Affecting Enzymes Activity:


Enzyme activity is affected by Nine Important factors: 

1): Enzyme Concentration

2): Substrate Concentration 

3): Product Concentration. 

4): Temperature 

5):Hydrogen Ion Concentration ( pH).

6): Poisons  and radiation 

7):Activators, 

8): Inhibitors:

9): Enzyme__ Substrate Complex.

1): Enzyme Concentration:


The rate of an enzyme__ catalysed reaction steadily increases with an increase in the number or enzyme molecules till a saturated effect is attained .




2): Substrate Concentration:


If there are more enzyme molecules than substrate molecules, a progressive increase in the substrate molecules ( S) increases the velocity ( V) of their conversion to products . However, eventually the rate of reaction reaches a maximum. At this stage, the active sites of all the available enzyme  molecules are occupied by the substrate molecules. Therefore, the substrate molecules occupy the active sites vacated by the products, and cannot increase the rate of reaction further.




Michaelis__ Menten constant


In an enzymatic reaction roman ➡️➡️P, the rate of the reaction is dependent on the substrate concentration. At low substrate concentration, the rate of reaction ' v' is proportional  to the substrate concentration. However, as the substrate concentration is increased,  the velocity of reaction falls and it is no longer proportional to the substrate concentration. With the further increase in substrate concentration, the rate of reaction becomes constant and independent of substrate concentration. The enzyme at this stage shows the saturation effect i.e., it has become saturated with the substrate. 




   This saturation effect led Michaelis and Menten to a  general theory of enzyme action and kinetics in the year 1913. Accordingly, the enzyme (E) first react with the substrate  (S) to form the enzyme substrate complex ( ES) which then breaks down to form free enzyme and the product ( P).
                  E2          K3
         E➕S ➡️➡️ES➡️➡️E➕P                  ...(1)
                  ⬅️⬅️     ⬅️⬅️
                    E1         K4
Both the steps are reversible and K1, K2, K3 and K4 are specific rate constants for the said reactions. To derive Michaelis__ Menten equation, rate of formation of ES and its breakdown is considered. 

   The rate of formation of ES from Eis given by___ 
      
             d [ES]
            _______= K1  ([E)  ➖[ES]) [S]        ...(2)
              dt

The rate of formation of ES from E➕P is negligible. 
Similarly, the rate of breakdown carbohydrates of ES is given by__

            ➖ d [ES]
            _______= K1  ([E)  ➕[ES]) [S]        ...(3)
               dt

When the reaction is in a steady state i.e., when the rate of formation of ES is equal to its rate of breakdown. With the ES concentration being constant, then

            K1([ES])[S]=K2[ES]➕K3[ES]       ...(4)
or    
           [S]([E]➖[ES])    K2➕K3
           _______________=__________=Km
                      [ES]            K1


The combined constant Km which is equal to K2+K
_______is called the Michaelis_ Menten               K1              constant.

The steady state concentration of ES may be obtained as

                 [E][S]    
 [ES] =   _________                                    ....(5)
               Km➕[S]
Since initial rate of an enzymatic reaction  (v) is proportional to the concentration of ES complex, therefore

                     v= k3[ ES]                           ....(6)

when substrate concentration is extremely high then whole of enzyme become saturated with substrate and exists as ESS complex. At.this state, maximum velocity of reaction ( Vmax) is reached
  
          Vmax= K3[ E]                                   .....(7)

When [E] represent total concentration of enzyme. Substituting value of ESfrom equation 5 into equation 6, we get

                        [E] [S]
            v=K3  _______                                   ....(8)  
                      Km+[S]

Dividing equation 8 by equation  7, we get

                           [E][S]
                     K3________
         V               Km+ [S]
     _____=    ______________
     Vmax          K3[E]
                                                     Vmax[S]
    By solving it, we obtain V=______
                                                     Km+[S]
      
    This is Michaelis _ Menten equation.

When v= ½Vmax , then ½Vmax= Vmax[S]
                                                                 ________
                                                                Km+ [S]
Diving this by Vmax we get

              [S]  
 ½= _______    or Km+ [S] =2[S] or Km=[S]
      Km+[S]     


Thus, Km is equal to the substrate concentration at which the velocity of the reaction is half maximum
The value of the Michaelis Menten constant is inversely proportional to the enzyme activity. A large value of Km means that a high substrate concentration is needed to get half velocity of the maximum rate of reaction.In true sense it means than enzyme has lower affinity for the substrate.


  When reciprocal values of enzyme activity and substrate concentration are potted against each other, we get a straight line. This double reciprocal plot is called the Line Weaver Burk Plot.From this plot, value of Km can be obtained only by  extending the line towards the abscissa.




3): Preoduct Concentration:

Accumulation of the product of enzyme reaction lowers the enzyme activity. Enzyme molecules must be freed to combine with more substrate molecules. Normally the products are quickly removed from the site of formation and the reaction does not suffer.

4): Temperature:

As temperature rises, the effectiveness of enzyme increases upto a certain optimum but decreases thereafter. Most enzymes show maximum activity in a temperature range of about 25 to 40°C. The optimum temperature for an enzyme usually corresponds to the body temperature of the organisms. Enzyme activity stops altogether at about 70 to 80°C and also at 0°C.




   Rise in temperature increases the kinetic energy of the molecules. Therefore, at higher temperature an increasing number of  molecules have the required activation energy and can take part in chemical reactions. At higher temperatures, the kinetic activity of molecules in an enzyms becomes strong enough to break the weak  hydrogen bonds that maintain the tertiary structure of the enzyme.Modifaction in the physical form of the enzyme results in the loss of its catalytic activity.This change in structure is called denaturation of protein. This is a permanent change, and the denatured enzyme protein remains inactive even if the temperature is then brought down. The enzymes are not destroyed by freezing, and regain their lost activity if the temperature is raised to normal.

      Freezing preserves the foods for a long time because neither the microbial enzymes nor the enzymes in the food can act at low temperature to spoil them. Cooking also preserves the food by destroying the enzymatic activity of bacteria that cause decay.

   The poikilothermous animals cannot live in very high and very low temperature, which greatly slow down enzyme action. This is why frog hibernates in winter and aestivates in host summer.


   Enzymes differ in their sensitivity to temperature. Bacteria living in host springs with temperature near 100°C have enzymes which can resist high temperatures. On the other hand, the cellular proteinscellular proteins of the plants and cold__ blooded animals of the arctic denature readily at moderate temperatures. Certain cyanobacteria live on the surface of glacial ice where temperature is close to the freezing point of water. Thus, the organisms have biochemical adaptability to environment. This has been achieved through long evolution. 

5): Hydrogen Ion Concentration ( pH) :

Some enzymes act best in an acid medium, other in an others in an alkaline medium. For every enzyme there is an optimum pH where its action is maximum. Most enzymes show maximum activity in a pH range of about 6.0 to 7.5, i.e., near neutral pH. A shift to the alkaline or acid side rapidly decreases the enzyme activity, and finally stops it altogether. This is due to denaturation of the enzyme molecules, i.e., change in its physical structure. Some digestive enzymes have their optimum in the acidic or alkaline range. For  example, pepsin of gastric juice has its optimum at pH2 ( acidic), and trypsin of pancreatic juice shows maximum activity at pH 8.8 ( alkaline).




  The H+ ions combine with negatively R groups on the enzyme.This electrically neutralizes the R groups and disrupt ionic bonds in the enzyme's folding pattern, thus changing its shape.


 6): Poisons and Radiation:

Poisons, such as cyanide, and radiation destroy the tertiary structure of the enzymes, making them ineffective. 

 7): Activators:

Certain enzymes are produced by the living cells in an inactivate ( nonfunctional) form. They are called the zymogens , or proenzymes. They are activated ( made functional) usually by hydrolysis of an inhibiting fragment that masks an active site, This is caused by specific ions or by other enzymes. Such ions and enzymes are called activators.

     Pepsinogen and trypsinogen are zymogens produced by  gastric glands and pancreas respectively. Pepsinogen is changed to active pepsin by hydrogen ions in the stomach. Trypsinogen is activated to trypsin by an enzyme enterokinase in the small intestine. Once small amount of pepsin or trypsin is formed, it itself  catalyzes the activation of remaining proenzymes. This process is called autocatalytic reaction, or autocatalysis.

8) Inhibitors: 

To somehow reduce or stop the action of an enzyme is called inhibition. Inhibition of enzyme action by denaturation has already been mentioned. Certain chemicals also limit or prevent the function of an enzyme. These are  called Inhibitors. They act in three different ways__


  (i) Competitive Inhibition:

This is brought about by a substance which closely resembles the substrate in molecular structure. Such a substance is called a competitive Inhibitor or substrate analogue.Because of its  close structural similarity to the substrate, the inhibitor competes with the substrate for the active sites of the enzyme, forming enzyme _ inhibitor complex instead of enzyme_ substrate complex. The enzyme _ inhibitor complex does not further enzyme activity. This is like jamming of a look by a key similar to the real one. A specific example of competitive Inhibition is cited. Malonate closely resembles succinate in structure and inhibits the action of succinate dehydrogenase. 



Use : The competitive inhibitors are used in the control of pathogenic bacteria. Sulphate drugs act as competitive inhibitors in the synthesis of folic acid in the bacterial cells because they compete with p_ amino benzoic acid for the active sites of the enzyme and check the synthesis.


Reversibility:

The competitive inhibition of enzyme activity is a reversible reaction. Excess of the substrate dislodges the inhibitor molecules, making the enzymes active again.

 Significanc:

Competitive inhibition is important because (i) it support the lock_ and _ key hypothesis of enzyme action, and 

(ii) It shows that substances structurally similar to substrates are not metabolised  by enzymes. 


(ii) Noncompetitive Inhibition: 

This is brought about by a substance which does not resemble the substrate in structure. The noncompetitive inhibitor binds to the enzyme af some site other than the substrate _ binding site, and no product is formed. Cyanide inhibits the mitochondrial enzyme cytochrome oxidase which is essential for cellular respiration. This kills the animals.
   

Many antibiotics are inhibitor of specific enzymes in bacteria. For instance, penicillin blocks the active site of an enzyme that many bacteria use to make their cell wall. DDT and parathion are inhibitors  of key enzymes in the nervous system.

Irreversibility 

The noncompetitive inhibition of an enzyme is irreversible. Addition of a substrate does not restore activity. 

(iii) Allosteric Inhibition: 

Still other inhibitors join an enzyme at a specific site and change the form of the active site meant for the substrate . These inhibitors are know  as modifiers, or modulators, and the sites where they fit in are called allosteric sites.Change of active site form prevents the binding of substrate to the enzyme and stop the reaction. The process is called allostery, or allosteric inhibition .The enzymes with allosteric sites are called allosteric enzymes. 



 A specific case of allosteric enzyme inhibition is cited. Glucose is changed to glucose 6_ phosphate in glycolysis with the help of the enzyme hexokinase. Glucose 6_ phosphate cause allosteric inhibition of hexokinase. This is called feedback allosteric Inhibition.




(9) : Enzyme_ Substrate Complex:

Enzyme_ substrate complex is formed in biocatalysis.The greater the affinity of the enzymeenzyme for its substrate , the greater is its catalytic action.















































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