Why does pepsin not denature at a low pH?


Why do some enzymes, like pepsin, not denature at a low pH?



  • Lowering pH does not denature many proteins. Unfolding, yes; denaturation, no.
  • Hydrogen bonds play an important role in stabilizing pepsin. Lowering pH does not necessarily break the hydrogen bond network.
  • At least one aspartyl residue in the catalytic site needs to be protonated for catalytic action, hence the function at low pH.
  • We do not completely understand stability of aspartly proteases, or pepsin.

Great question, let’s talk enzymes!

First off, activity and stability are two very different properties. When you talk about *denaturation*, you are referring to stability,  and *activity* refers to function.  An enzyme is stable within a range of pH (and temperature), but stability need not result in complete (or any) activity.

Pepsin is most active between pH 1.5-2.5, but it is stable between 1-7.5 pH units. Between pH 5/6 to 7.5, the enzyme is stable but exhibits ZERO activity. However, you can decrease the pH to 2 in this state and regain back full activity.  Image from : Page on bmj.com

So, I am going to break your question into two:

  1. How does pH affect stability of pepsin?
  2. Why does pepsin need  H+ ions to function?

Proteases in general are difficult to study, the active site is usually mutated and mutants are studied. This also means that we don’t know too much about proteases in general, let alone the ones active at lower pH.

How does pH affect stability of pepsin?

  • Proteins are stable over a range of pH, and this range is specific to each protein and dependent on charged interactions/charge distributions/salt bridges/disulphide interactions etc.
  • When pH is low the charged groups are protonated, which might in turn disrupt interactions present in the native state of the protein.
  • So, technically, the driving force for denaturation is electrostatic repulsion.
  • However, this repulsion does not always win over other forces, such as hydrophobicity, or disulphide bridges.
  • In fact there are many proteins that maintain their native state at pH as low as 0.5.  Some proteins also unfold a little and refold back, with decrease in pH (often referred to as the A state/molten globule). [Page on 237.112.247]].
  • Here is a completely misleading representation, as a folded state is driven by a number of interactions.

Image from : Rules of Protein Structure

  • Many enzymes retain structure at low pH, these include  carbonic anhydrase, beta-Iactamase, staphylococcal nuclease, ribonuclease, chymotrypsinogen. [Annual Review of Biochemistry]
  • In the case of pepsin, there is some amount of unfolding at lower pH. This exposes the catalytic site, thus making it functional, (i.e cleaving of the 44 residue N terminal prosegment from the zymogen). This is true for many aspartyl proteases. [Dynamics of Thermodynamically Stable, Kinetically Trapped, and Inhibitor-Bound States of Pepsin]
  • Pepsin also has fewer basic residues, hence lesser positive charge to drive electrostatic repulsions.

Image from : Page on nih.gov

  • However, we don’t know too much about stability, for example :what makes it stable at low pH? what interactions change with change in pH? Does the hydrophobic pocket  remain unaffected? if yes, why? what is the effect of temperature? what are the effects of ions at low pH? what is the role of conformational entropy? and so on.

Further reading:
Classification of Acid Denaturation of Proteins: Intermediates and Unfolded States
Annual Review of Biochemistry

Why does pepsin need  H+ ions to function?

  • Pepsin is active at acidic pH since the catalytically competent enzyme has one protonated (Asp 32) and one deprotonated Asp (Asp 215) at the active site. This state is typically achieved at an acidic pH, and is consistent for many aspartyl proteases [ Binding of a reduced peptide inhibitor to the aspartic proteinase f…The structure and function of the aspartic proteinases. ]
  • Asp 215 is know to be involved in a H-bond with a neighboring threonine and Asp 32, protonated. This distinction, and the protonation of Asp32 is known to be critical for substrate binding and subsequently, catalysis.
  • So, yes, the protein *is* catalytically active only in the presence of H+ ions.

Thr 218 hydroxyl takes up a guard position near the Asp 215 carboxyl at a distance of a hydrogen bond, as in porcine pepsin. This hydrogen bond utilizes the anti lone pair electrons of the outer oδ2 oxygen, while the syn lone pair of this oxygen is engaged in the hydrogen bond with the water molecule W1. As a result, the ability of the outer oxygen of Asp 215 to bind protons from bulky solvent becomes rather low. At the same time, the distance between the Ser 35 Oγ atom and Asp 32 carboxyl is too large for the formation of a hydrogen bond. From : Page on nih.gov

For further reading:
Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease
Overview of pepsin-like aspartic peptidases.
Page on www.enzim.hu

From Quora


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