Effect of pH on enzyme activity

The optimal pH for pepsin is 2, reflecting its need as a digestive enzyme in the gastric juice of the stomach; whereas the optimal pH for alkaline phosphatase is 9, reflecting the basic environment in bone (Figure-1)

Effect of pH


Figure-1- Effect of pH on catalytic activity of enzyme.

Changes in the pH can alter these changes, so that the reaction proceeds at a slower rate. If the pH is too high or too low, the enzyme can also undergo denaturation. Which of the following statements is true of denaturation?

A. The enzyme loses its capacity to hold the substrate

B. The 3 D structure of the enzyme is disrupted

C. The active site is lost

D. The enzyme fails to bind the coenzyme

E. All of the above.

The correct answer is- E.

Basic concept

Proteins perform complex physical and catalytic functions by positioning specific chemical groups in a precise three-dimensional arrangement.

Protein structure can be described at four levels. The primary structure refers to the amino acid sequence. The secondary structure refers to the conformation adopted by local regions of the polypeptide chain.

Tertiary structure describes the overall folding of the polypeptide chain. It refers to the entire three-dimensional conformation of a polypeptide indicating, in three-dimensional space, how secondary structural features—helices, sheets, bends, turns, and loops—assemble to form domains and how these domains relate spatially to one another.

Finally, quaternary structure refers to the specific association of multiple polypeptide chains to form multi subunit complexes.

A protein spontaneously folds into a well-defined and elaborate three-dimensional structure that is dictated entirely by the sequence of amino acids along its chain (Figure -2).The self-folding nature of proteins constitutes the transition from the one-dimensional world of sequence information to the three-dimensional world of biological function.

Protein folding

Figure-2- Protein folding-the three-dimensional structure of a protein, a linear polymer of amino acids, is dictated by its amino acid sequence.

Higher orders of protein structure are stabilized primarily—and often exclusively—by noncovalent interactions.

Principal among these are hydrophobic interactions that drive most hydrophobic amino acid side chains into the interior of the protein, shielding them from water. Other significant contributors include hydrogen bonds and salt bridges between the carboxylates of aspartic and glutamic acid and the oppositely charged side chains of protonated lysyl, argininyl, and histidyl residues. Some proteins contain covalent disulfide (S—S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen. Intra polypeptide disulfide bonds further enhance the stability of the folded conformation of a peptide, while interpolypeptide disulfide bonds stabilize the quaternary structure of certain oligomeric proteins.


Denaturation involves protein unfolding, i.e., disruption of higher orders of protein structure, secondary, tertiary and quaternary structure (if present), the primary structure remains intact. The proteins can be denatured by heat, mechanical pressure or by chemical denaturants. Basically by these agents, the non covalent interactions  that stabilize the higher orders of organization are broken, resulting in unfolding of the polypeptide chain.

A protein loses its functional capacity upon denaturation, showing thereby a close structure-function relationship. Denaturation can be reversible or irreversible. Heating or boiling of a protein results in irreversible denaturation.

Effect of denaturation on enzymatic activity

Let us consider Ribonuclease as a representative enzyme. The functional form of the enzyme is maintained mainly by disulphide bridges, (figure-3), though other non covalent interactions also participate in supporting the tertiary structure.

In the presence of Urea and β- Mercaptoethanol (figure-23), the enzyme undergoes denaturation to form a scrambled structure. In that structure, the enzyme loses its overall catalytic capacity.


Denaturation of ribonuclease

Figure-3- Denaturation of Ribonuclease by reduction- the disulphide bridges are broken (between 26 and 84; 40 and 95; 58 and 110; and between 65 and 72) in the presence of reducing agents.

This denaturation is reversible, if the denaturating agents are removed, the disulphide bridges are reformed and the enzyme regains its native 3 dimensional functional form (figure-4).


Renaturation of ribonuclease

Figure-4- Native Ribonuclease can be reformed from scrambled Ribonuclease in the absence of urea but in presence of a trace of β-Mercaptoethanol. The disulphide bridges are re-established and the native conformation is regained.

As regards options given in the question, every options seems to be correct-

A. The enzyme loses its capacity to hold the substrate- The unfolded enzyme fails to bind the substrate. This option could have been the right answer.

B. The 3 D structure of the enzyme is disrupted- Very true, denaturation leads to disruption of 3 D structure only; it can be the best option.

C. The active site is lost- very much true. If the 3D structure is lost upon denaturation, the active site is also lost, also the correct option.

D. The enzyme fails to bind the coenzyme- Again the same logic. The binding of the co enzyme takes place at a special site, and that is attained by acquiring the 3-D structure. Disruption of 3-D or tertiary structure leads to loss of capacity to bind the coenzyme also. This is also correct of denaturation.

E. All of the above – Considering all options, E -is the most suited option, because of the processes affected upon denaturation.

Had there been other options, not affected by denaturation, the option B- Disruption of 3-D structure could have been the best answer.


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