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I read chapter 9 in the book Biochemistry (5th edition), by Berg, Tymoczko, and Stryer (provided in the NCBI site here). It describes the mechanism of action of the chymotrypsin enzyme. The catalysis is performed through the catalytic triad consisting of serine-195, histidine-57, and aspartate-102.

What intrigued me is the following section on the related enzyme subtilisin, which also has a catalytic triad but with different residue numbers:

9.1.5. The Catalytic Triad Has Been Dissected by Site-Directed Mutagenesis

[...] As expected, the conversion of active-site serine 221 into alanine dramatically reduced catalytic power. [...] The mutation of histidine 64 to alanine had very similar effects. [...]

These observations support the notion that the serine-histidine pair act together to generate a nucleophile of sufficient power to attack the carbonyl group of a peptide bond. The conversion of aspartate 32 into alanine had a smaller effect, although the value of $k_\mathrm{cat}$ still fell to less than 0.005% of its wild-type value.

My problem is in what it says next:

The simultaneously conversion of all three catalytic triad residues into alanine was no more deleterious than the conversion of serine or histidine alone. Despite the reduction in their catalytic power, the mutated enzymes still hydrolyze peptides a thousand times as rapidly as does buffer at pH 8.6.

A graph of the relative rates can be taken from this link:

Reaction rates with wild-type and various mutants

My question is: how can it be that after replacing all the triad residues, the enzyme still possess such a great catalytic activity? What is the mechanism behind that phenomenon?

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If the substrate still binds to the protein, other parts of the mechanism are still in place. Specifically, the main chain amide groups that help to stabilize the tetrahedral intermediate are still present. In the absence of serine, water will attack the carbonyl. In the presence of serine and the absence of the histidine, serine will still be the nucleophile, but a less efficient one.

Look up oxyanion hole if this part of the mechanism is unfamiliar to you. enter image description here

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  • $\begingroup$ yea, but water can attack the substrate protein even without the enzyme. why the big increase in catalysis compared to basic solution? $\endgroup$ – Ytfu Gjuf Feb 12 at 21:35
  • $\begingroup$ i'll explain my question further: water is a small molecule and it's abundant in the solution so it doesn't seems that the substrate need to be held in a particular position in order to be attacked by water. how can the oxyanion hole can perfect the catalysis if the water molecule is available in place even without it's help. thank you! $\endgroup$ – Ytfu Gjuf Feb 13 at 17:21
  • $\begingroup$ @YtfuGjuf The oxyanion hole makes the substrate a better electrophile, just like the Asp and His in the triad make Ser (and the water in the second part of the mechanism) a better nucleophile. The substrate has to be in a particular position so that activation of the electrophile (the carbonyl) works. $\endgroup$ – Karsten Theis Feb 13 at 18:51

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