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I can imagine that protonated amino acid form, particularly at the active site, is important for the catalytic activity so hydrogen bonds can be created between the substrate and the enzyme. However, I cannot imagine how the ionized form can be important for the activity?

Both forms are important for the activity as stated in this study:

https://www.ncbi.nlm.nih.gov/pubmed/7306491

The pH dependence of log V/K for dihydrofolate showed that a group with a pK value of 4.7 must be ionized and that a group with a pK value of 6.6 must be protonated for activity

I cannot understand how the ionized form is important for the activity, could some one help me to understand that?

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  • $\begingroup$ Without seeing a crystal structure it's hard to tell. But the abstract suggests 2 carboxyl groups, on a glutamate and aspartate. The abstract also mentions a lysine is necessary to maintain active conformation. Could the negative carboxyl group form an ionic bond with a postively charged lysine? $\endgroup$ – user137 Nov 28 '17 at 2:47
  • $\begingroup$ @user137, I am not sure, but isn't the ionic bond too strong for enzyme-substrate complex? $\endgroup$ – Mohammed Noureldin Nov 28 '17 at 15:34
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Enzymatic reactions are chemical reactions. Chemical reactions involve charge ("electron") transfer. Charge transfer occurs more readily if there is a large gradient in charge density between the attacking active site group and the attacked atom of the substrate. Many enzymes (yes, it is really quite common) use ionic forms of aspartate or glutamate as a catalytic amino acid residue.

For example, some enzymes belonging to the alpha/beta-hydrolase fold family (e.g., haloalkane dehalogenases, epoxide hydrolases) use a deprotonated aspartate as the nucleophile (which attacks an electrophilic atom of the substrate); the specific position of the nucleophile within the enzyme and its interaction with other groups in the enzyme enhance the nucleophilic nature of the aspartate, a phenomenon called "oxyanion hole."


Ollis et al. (1992) Protein Eng 5: 197-211. https://www.ncbi.nlm.nih.gov/pubmed/1409539

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Although the answer provided by @MartinKlvana is correct, I would like to clarify the question of the strengths of non-covalent interactions in proteins, as this seems to be the basis of the poster’s problem. As I would put it:

Life is dynamic. So the chemistry of life depends on weak interactions that can be made and broken.

Ionic interactions are similar in energy to other non-covalent interactions

According to Berg et al. and the Wikipedia article typical ranges for non-covalent interactions are:

  • Ionic interaction: 1.4 kcal/mol (3Å in water — depends on distance and dielectric constant)
  • Hydrogen bond: 1–3 kcal/mol (depends on distance and angle)
  • Van der Walls interaction: 0.5–1.0 kcal/mol (depends on distance)

This is in contrast to, say the covalent C–C bond with an energy of about 100 kcal/mol according to this site. Thus, the ionic bond is clearly a relatively weak bond.

The charged state of a residue involved in enzyme mechanisms is often partial and dynamic

As the reader will know, the percentage ionization of a residue depends on its pKa and that of the medium. In neutral solution a histidine side chain (pKa 6.0) would be only 10% protonated. At the active site of a protein things may be different however. In a hydrophobic environment the acidic side chains of aspartic or glutamic acid may have far less tendency to ionize, and thus have an effective pKa much higher than that in water (4.1). The proximity of other residues may also influence the ionization or protonation by accepting or donating a hydrogen ion.

The point of this is that the residues involved in catalysis are often effectively less charged than a fully charged residue in solution, and this allows the ionization or protonation to be reversed at the end of the reaction, returning the enzyme catalyst to its original state.

Some examples

Although the functional role of Asp-27 (the presumed pKa 4.7 group) in tetrahydrofolate reductase would appear to be to donate a proton to the substrate, the details of this reaction are not completely clear. So it is easier to illustrate this point with two classic enzymes.

Serine Proteases (Trypsin etc)

More details of this can be found on the web and in any biochemical text, but the following illustration taken from Berg et al. illustrates the key point I wish to make.

Catalytic triad in serine proteases

This is that during the reaction the histidine is able to alternate between being protonated and unprotonated, receiving a proton from the adjacent serine.

Lysozyme

Here the mechanism of action involves two acidic groups, one charged (Asp-52) and one (Glu-35) uncharged. This difference is because the latter is in a hydrophobic environment. During the course of the reaction however it is able to ionize, but returns to its protonated form when the reaction is complete. This is shown in the illustration below, taken from a Nature Structural Biology review article by A.J. Kirby.

mechanism of action of lysozyme

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