Subtilisin
{{Short description|Proteolytic enzyme found in Bacillus subtilis}}
{{Pfam box
| Name = Peptidase S8, subtilisin-related
| Symbol = Peptidase_S8
| image = File:PDB 2pmw EBI.png
| caption = S8 + I9 (lower-right), Bacillus subtilis ({{PDB|2pmw}})
| Pfam = PF00082
| InterPro = IPR015500
| CDD = cd07477
| PROSITE = PDOC00125
| SCOP = 1cse
| CATH = 1cse
}}
{{Infobox nonhuman protein
| Organism = Bacillus amyloliquefaciens
| Name = Subtilisin BPN'
| image = Crystal structure of Subtilisin - 1st2.png
| caption = Crystal structure of subtilisin S8 domain.{{PDB|1st2}}; {{cite journal | vauthors = Bott R, Ultsch M, Kossiakoff A, Graycar T, Katz B, Power S | title = The three-dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8 A and an analysis of the structural consequences of peroxide inactivation | journal = The Journal of Biological Chemistry | volume = 263 | issue = 16 | pages = 7895–906 | date = June 1988 | doi = 10.1016/S0021-9258(18)68582-5 | pmid = 3286644 | doi-access = free }}
| Symbol = apr
| AltSymbols =
| IUPHAR_id =
| ATC_prefix =
| ATC_suffix =
| ATC_supplemental =
| CAS_number = 9014-01-1
| CAS_supplemental =
| DrugBank =
| EntrezGene = 5712479
| HGNCid =
| OMIM =
| PDB = 1st2
| PDB_supplemental = [http://www.ebi.ac.uk/pdbe/searchResults.html?display=pdb&search_type=advanced&sort_column=reverse_release_date&Uniprot=P00782&term=UniProt:%20P00782 More structures]
| RefSeq =
| UniProt = P00782
| ECnumber = 3.4.21.62
| footnotes= GO:0004252
}}
Subtilisin is a protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis.{{cite book | title = The subtilisins |vauthors = Ottesen M, Svendsen I | series = Methods Enzymol. | year = 1970 | volume = 19 | pages = 199–215 | doi=10.1016/0076-6879(70)19014-8|isbn = 978-0-12-181881-4 }}{{cite book | chapter = Subtilisins: primary structure, chemical and physical properties | title = The Enzymes |vauthors = Markland FS, Smith EL |date = 1971 |volume = 3 |pages = 561–608 |editor = Boyer PD |edition = 3rd |publisher = Academic Press |location = New York }}{{cite journal | vauthors = Philipp M, Bender ML | title = Kinetics of subtilisin and thiolsubtilisin | journal = Molecular and Cellular Biochemistry | volume = 51 | issue = 1 | pages = 5–32 | year = 1983 | pmid = 6343835 | doi = 10.1007/bf00215583 | s2cid = 24136200 }}{{cite journal | vauthors = Nedkov P, Oberthür W, Braunitzer G | title = Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus | journal = Biological Chemistry Hoppe-Seyler | volume = 366 | issue = 4 | pages = 421–30 | date = April 1985 | pmid = 3927935 | doi = 10.1515/bchm3.1985.366.1.421 }}{{cite journal | vauthors = Ikemura H, Takagi H, Inouye M | title = Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli | journal = The Journal of Biological Chemistry | volume = 262 | issue = 16 | pages = 7859–64 | date = June 1987 | doi = 10.1016/S0021-9258(18)47646-6 | pmid = 3108260 | doi-access = free }}{{cite book | veditors = Brocklehurst K, Neuberger A | title = Hydrolytic enzymes | publisher = Elsevier | location = Amsterdam | year = 1987 | isbn = 0-444-80886-8 | chapter = Structure and function of serine proteases | vauthors = Polgár L }}{{cite journal | vauthors = Vasantha N, Thompson LD, Rhodes C, Banner C, Nagle J, Filpula D | title = Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein | journal = Journal of Bacteriology | volume = 159 | issue = 3 | pages = 811–9 | date = September 1984 | pmid = 6090391 | pmc = 215730 | doi = 10.1128/JB.159.3.811-819.1984 }}
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 27kDa. They can be obtained from certain types of soil bacteria, for example, Bacillus amyloliquefaciens from which they are secreted in large amounts.
Nomenclature
"Subtilisin" does not refer to a single protein, but to an entire clade under subtilases containing the classical subtilisins. The clade can be further divided into four groups: "true subtilisins" (containing the classical members), "high-alkaline subtilisins", "intracellular subtilisins", and "phylogenetically intermediate subtilisins" (PIS).{{cite journal |last1=Falkenberg |first1=Fabian |last2=Rahba |first2=Jade |last3=Fischer |first3=David |last4=Bott |first4=Michael |last5=Bongaerts |first5=Johannes |last6=Siegert |first6=Petra |title=Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101 T |journal=FEBS Open Bio |date=October 2022 |volume=12 |issue=10 |pages=1729–1746 |doi=10.1002/2211-5463.13457|pmid=35727859 |pmc=9527586 }}{{cite journal |last1=Falkenberg |first1=F |last2=Bott |first2=M |last3=Bongaerts |first3=J |last4=Siegert |first4=P |title=Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae. |journal=Frontiers in Microbiology |date=2022 |volume=13 |pages=1017978 |doi=10.3389/fmicb.2022.1017978 |doi-access=free |pmid=36225363 |pmc=9549277}} Notable subtilisins include:
Other non-commercial names include ALK-enzyme, bacillopeptidase, Bacillus subtilis alkaline proteinase, colistinase, genenase I, protease XXVII, subtilopeptidase, kazusase, protease VIII, protin A 3L, protease S.
Other commercial names with unidentified molecular identities include SP 266, orientase 10B (HBI Enzymes), Progress (Novozyme), Liquanase (Novozyme).
Structure
The structure of subtilisin has been determined by X-ray crystallography. The mature form is a 275-residue globular protein with several alpha-helices, and a large beta-sheet. The N-terminal contains an I9 propeptide 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.
Mechanism of catalysis
{{Unsourced|section|date=June 2024}}
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.
Applications
= Research tool =
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.
= Commercial =
Protein-engineered subtilisins are widely used in commercial products (the native enzyme is easily inactivated by detergents and high temperatures) and is also called a stain cutter, for example, in laundry{{cite web | title = Spar Washing Detergent contents | url = http://www.5010358128009.detergent-info.com/ }} and dishwashing detergents, cosmetics, food processing,{{cite web |last1=Chaplin |first1=Martin | name-list-style = vanc |title=Applications of proteases in the food industry |url= http://www1.lsbu.ac.uk/biology/enztech/proteases.html |website= London South Bank University |access-date=3 March 2015 |archive-url= https://web.archive.org/web/20100314220833/http://www1.lsbu.ac.uk/biology/enztech/proteases.html |archive-date=2010-03-14 |date=20 December 2004}} skin care products, contact lens cleaners, and for research in synthetic organic chemistry.
= Occupational safety and health =
People can be exposed to subtilisin in the workplace by breathing it in, swallowing it, skin contact, and eye contact. The National Institute for Occupational Safety and Health (NIOSH) has set a recommended exposure limit (REL) of 60 ng/m3 over a 60-minute period.{{cite web |title = CDC - NIOSH Pocket Guide to Chemical Hazards - Subtilisins |url = https://www.cdc.gov/niosh/npg/npgd0572.html |website = www.cdc.gov |access-date = 2015-11-21 }}
Subtilisin can cause "enzymatic detergent asthma". People who are sensitive to Subtilisin (Alcalase) usually are also allergic to the bacterium Bacillus subtilis. Mosby's Medical, Nursing, & Allied Health Dictionary, 14th edition, page 557
See also
References
{{Reflist|35em}}
{{Serine endopeptidases}}
{{Enzymes}}
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