The side-chain of the nucleophilic residue performs covalent catalysis on the substrate. The lone pair of electrons present on the oxygen or sulfur attacks the electropositive carbonyl carbon. The 20 naturally occurring biological amino acids do not contain any sufficiently nucleophilic functional groups for many difficult catalytic reactions. Embedding the nucleophile in a triad increases its reactivity for efficient catalysis. The most commonly used nucleophiles are the hydroxyl (OH) of serine and the thiol/thiolate ion (SH/S) of cysteine. Alternatively, threonine proteases use the secondary hydroxyl of threonine, however due to steric hindrance of the side chains extra methyl group such proteases use their N'-terminal amide as the base, rather than a separate amino acid.
Use of oxygen or sulfur as the nucleophilic atom causes minor differences in catalysis. Compared to oxygen, sulfurs extra d orbital makes it larger (by 0.4 Å) and softer, allows it to form longer bonds (d and d by 1.3-fold), and gives it a lower pK (by 5 units). Serine is therefore more dependent than cysteine on optimal orientation of the acid-base triad members to reduce its pK in order to achieve concerted deprotonation with catalysis. The low pK' of cysteine works to its disadvantage in the resolution of the first tetrahedral intermediate as unproductive reversal of the original nucleophilic attack is the more favourable breakdown product. The triad base is therefore preferentially oriented to protonate the leaving group amide to ensure that it is ejected to leave the enzyme sulfur covalently bound to the substrate N-terminus. Finally, resolution of the acyl-enzyme (to release the substrate C-terminus) requires serine to be re-protonated whereas cysteine can leave as S. Sterically, the sulfur of cysteine also forms longer bonds and has a bulkier van der Waals radius and if mutated to serine can be trapped in unproductive orientations in the active site.
Very rarely, the selenium atom of the uncommon amino acid selenocysteine is used as a nucleophile. The deprotonated Se state is strongly favoured when in a catalytic triad.