Threonine proteases use the amino acid threonine as their catalytic nucleophile. Unlike cysteine and serine, threonine is a secondary hydroxyl (i.e. has a methyl group). This methyl group greatly restricts the possible orientations of triad and substrate as the methyl clashes with either the enzyme backbone or histidine base. When the nucleophile of a serine protease was mutated to threonine, the methyl occupied a mixture of positions, most of which prevented substrate binding. Consequently, the catalytic residue of a threonine protease is located at its N-terminus.
Two evolutionarily independent enzyme superfamilies with different protein folds are known to use the N-terminal residue as a nucleophile: Superfamily PB (proteasomes using the Ntn fold) and Superfamily PE (acetyltransferases using the DOM fold) This commonality of active site structure in completely different protein folds indicates that the active site evolved convergently in those superfamilies.
Families of threonine proteases