Subtilisin

Unknown parameter footnote Unknown parameter footnote Subtilisin is a non-specific protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis. Subtilisins belong to subtilases, a group of serine proteases that – like all serine proteases – initiate the nucleophilic attack on the peptide (amide) bond through a serine residue at the active site. Subtilisins typically have molecular weights of about 20,000 to 45,000 dalton. They can be obtained from certain types of soil bacteria, for example, Bacillus amyloliquefaciens from which they are secreted in large amounts. Subtilisin is also commercially known as Alcalase®, Alcalase® 0.6L, Alcalase® 2.5L, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline proteinase bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, genenase I, Esperase®, maxatase, thermoase PC 10, protease XXVII, thermoase, superase, subtilisin DY, subtilopeptidase, SP 266, Savinase® 8.0L, Savinase® 4.0T, kazusase, protease VIII, opticlean, Bacillus subtilis alkaline proteinase, protin A 3L, Savinase®, Savinase® 16.0L, Savinase® 32.0 L EX, orientase 10B, protease S). It is the type serine endopeptidase of MEROPS family S8. The structure of subtilisin has been determined by X-ray crystallography. The mature form is a 247-residue globular protein with several alpha-helices, and a large beta-sheet. The N-terminal contains an I9 propetide domain (InterPro: IPR010259) that assists the folding of subtilisin. Proteolytic removal of the domain activates the enzyme. It is structurally unrelated to the chymotrypsin-clan of serine proteases, but uses the same type of catalytic triad in the active site. This makes it a classic example of convergent evolution. The active site features a charge-relay network involving Asp-32, His-64, and active site Ser-221 arranged in a catalytic triad. The charge-relay network functions as follows: The carboxylate side-chain of Asp-32 hydrogen-bonds to a nitrogen-bonded proton on His-64's imidazole ring. This is possible because Asp is negatively charged at physiological pH. The other nitrogen on His-64 hydrogen-bonds to the O-H proton of Ser-221. This last interaction results in charge-separation of O-H, with the oxygen atom being more nucleophilic. This allows the oxygen atom of Ser-221 to attack incoming substrates (i.e., peptide bonds), assisted by a neighboring carboxyamide side-chain of Asn-155. Even though Asp-32, His-64, and Ser-221 are sequentially far apart, they converge in the 3D structure to form the active site. To summarize the interactions described above, Ser-221 acts as a nucleophile and cleaves peptide bonds with its partially negative oxygen atom. This is possible due to the nature of the charge-relay site of subtilisin. In molecular biology using B. subtilis as a model organism, the gene encoding subtilisin (aprE) is often the second gene of choice after amyE for integrating reporter constructs into, due to its dispensability.