Subtilisin E is an alkaline serine protease found in Bacillus subtilis that has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease becomes effectively active. [5]

O-(2-Nitrobenzyl)-L-tyrosine (abbr.: ONBY) is a derivate of the canonical amino acid tyrosine. It carries a photo-labile protection group that can be cleaved off by irradiation with UV-light (365nm, [6]).

By adding ONBY to the genetic code of E. coli and incorporating said amino acid in the pro-peptide cleavage-site of subtilisin E the maturation process is disturbed. Due to its size ONBY sterically hinders the protease [7]. The pro-peptide cannot be cleaved from the enzyme and subtilisin E is not able to achieve its full proteolytic activity. A temporarily inactive protease is produced.