To begin examining the catalytic processes of the serine proteases, let's look at the reaction as it would occur in aqueous acid:

• Amides protonate on the carbonyl oxygen rather than the nitrogen, thus preserving the delocalization of the nitrogen lone pair.

• Among the evidence for this is the observation that rotational barriers of peptide bonds are increased in acid, whereas protonation on N should eliminate the barrier.

• Amides are the most difficult to hydrolyze of acyl derivatives because they have the greatest delocalization in the acyl group
  • The attack of nucleophilic water ends this delocalization

• Deprotonation forms a tetrahedral intermediate, which is common to all nucleophilic acyl substitution reactions

• In a simple amide, the two OH groups of the tetrahedral intermediate are enantiotopic
  • In a polypeptide with stereogenic centers, they are diastereotopic

• Protonation of the tetrahedral intermediate occurrs exclusively on N, since it is by several orders of magnitude the most basic site
  • Delocalization of the N unshared pair is no longer possible

• The driving force for the breakdown of the N-protonated species is the formation of the very strong C=O double bond.

Given that organic chemists have found ONLY this mechanism for hydrolysis of ALL acyl derivatives, we expect the enzyme mechanism to lookk much like this.

The catalytic work of the proteases is done by the so-called catalytic triad, Asp102, His57, and Ser195:

That these are the catalytic residues was established in a variety of ways, largely before site-directed mutation was devised.

• A number of covalent inhibitors were found. For example, tosyl phenylalanine chloromethyl ketone reacts irreversibly with His57 and inactivates the enzyme:
  

• Tosyl fluoride reacts with Ser195
  
  • A crystal structure of the inactivated enzyme shows the fit of the aromatic ring into its binding pocket:
  

• The aromatic side chain pocket is 10-12 A deep, and about 4 x 6.5 A in cross section
  • Benzene rings are about 6 A wide and 3.5 A thick.

Site-directed mutations have been done on subtilisin, which uses the same catalytic triad. The active site residues were replaced one at a time with alanine: