It is reported in many papers, that some residues in the active site of enzymes need to be protonated to get functional enzyme, where these residues have a low pKa (for let us say 5).

How can that happen in the physiological conditions (with pH=~7.4)?

It is mentioned that a raise in the pKa value is happening during the catalytic activity to get that protonation state. But how is that happening?

Here is a publication where this protonation and pKa raising is described.


This conclusion is confirmed by the result that a group with a pK value of 6.7 must be protonated for the binding of methotrexate. It is proposed that the binding involves the formation with N-5 of dihydrofolate or N-1 of methotrexate of a hydrogen bond which has considerable ionic character and which lies within a hydrophobic environment. Further, it is suggested that the same hydrogen acts as an auxiliary catalyst which facilitates hydride transfer from NADPH to dihydrofolate for its conversion to tetrahydrofolate. Evidence to support this suggestion comes from the finding that the V profile is similar to the V/K profile except that the pK of the group which must be protonated for maximum enzyme activity is shifted upward by about 2 pH units. Such an increase in a pK value is consistent with the formation of a hydrogen ionic bond in the ternary enzyme-NADPH-dihydrofolate complex. The results of inactivation experiments with trinitrobenzenesulfonate appear to indicate that a lysine residue is necessary to maintain the enzyme in its active conformation.


Generally speaking, even though physiological pH is generally ~7.4, this is not necessarily the pH inside the enzyme active site. The enzyme's catalytic machinery may experience a completely different pH for one of two reasons: 1) because the enzyme is located in a place where the pH is simply not 7.4, and 2) The enzyme's amino acid composition shifts either the pH in the active site or the pKa of the active site residues relative to the surrounding environment such that the effective local pH in the catalytic site is lower or higher than 7.4.

For example, there are places in humans, such as the stomach or around muscle cells during anaerobic respiration where the pH is actually acidic relative to the standard physiological pH of 7.4, due to the presence of HCl or lactic acid respectively. Additionally, certain subcellular organelles, such as lysosomes, also are relatively acidic, since they are a site for breaking down proteins into amino acids.

However, the case presented in this paper seems to be an example of the second situation, where an enzyme that is in a pH neutral environment relies on one or more protonated amino acids to perform catalysis. This is relatively common. Basically, the enzyme's active site has an amino acid composition that when folded correctly, alters the local pH around the catalytic residues to favor catalysis. For example, in the enzyme acetylcholinesterase, the active site serine is rendered nucleophilic by being positioned next to a glutamate and a histidine, in an arrangement referred to as a catalytic triad. The glutamate is an acidic residue, and its close proximity to the histidine means that it will stabilize the protonated form of the histidine. Energetically, this makes protonation of the histidine very favorable. This causes the histidine to take a proton from the active site serine. Now that serine's oxygen is deprotonated, it can act as a strong nucleophile. After the serine forms a covalent interaction with the substrate, the histidine will facilitate a similar nucleophilic attack by a water molecule, regenerating the enzyme and completing the catalytic cycle.

Something similar is seen with the beta-lactamase TEM-1. In this case, TEM-1 uses a lysine (pKa ~11) instead of histidine (pKa ~6), so multiple acidic residues are in the active site to facilitate the deprotonation of the lysine during the catalytic cycle. In effect, the amino acid composition of the active site allows for the shuffling of protons needed for catalysis.

So to summarize, while physiological pH is widely regarded to be 7.4, an enzyme's catalytic machinery may experience a very different pH through either the cellular/physiological localization, or through the amino acid composition of the active site. In the latter case, the presence of acidic or basic residues can alter the protonation state of other acidic or basic residues, meaning the effective pKa of these residues or the effective pH around them is higher or lower than 7.4.

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    $\begingroup$ Additionally, many enzymes use metal cofactors that facilitate proton transfer reactions by altering pKa of active site groups. For example, DNA polymerase beta binds two Mg2+ ions, one of which alters pKa of the 3'-OH group of the 3'-terminal nucleotide, which makes the proton transfer to an active site aspartate residue or to the bulk water thermodynamically (much more) feasible. $\endgroup$ – user37894 Nov 20 '17 at 10:44
  • $\begingroup$ It seems incorrect to say that the enzymes are altering the pH of their active sites. Rather they are altering the pKa of the active site residues. I would also add that pKa is a measure of equilibrium and thus even if, for example, the pKa of a moiety is below the pH, this does not mean it is completely deprotonated. $\endgroup$ – canadianer Nov 20 '17 at 19:00
  • $\begingroup$ Martin - Thanks, I totally forgot to mention that; it is an important point. $\endgroup$ – Hayden S Nov 21 '17 at 3:24
  • $\begingroup$ Canadianer - Thanks! Your second point is important and I didn't to mention it. Concerning your first point, while I agree that it is generally more useful to think about residue pKa rather than pH of the active site changing, and that this is probably the more important factor, I would think (I may be wrong) that both are changing. If a pocket in a protein has many acidic or basic residues, the solvent in the pocket will likely have more or less hydrogen ions per unit volume than the solvent outside the pocket. Since pH is a measure of [H+], this would differ between the two environments. $\endgroup$ – Hayden S Nov 21 '17 at 3:35

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