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Subtilisin is a non-specific protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis.^[2]^[3]^[4]^[5]^[6]^[7]

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.

Nomenclature

Subtilisin is also 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, serine endopeptidase).

Structure

The structure of subtilisin has been determined by X-ray crystallography. It is a 275-residue globular protein with several alpha-helices, and a large beta-sheet. 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

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^[8] and dishwashing detergents, cosmetics, food processing,^[9] skin care ointments,^[10] contact lens cleaners, and for research in synthetic organic chemistry.

References

^ PDB 1st2; Bott R, Ultsch M, Kossiakoff A, Graycar T, Katz B, Power S (June 1988). "The three-dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8 A and an analysis of the structural consequences of peroxide inactivation". J. Biol. Chem. 263 (16): 7895–906. PMID 3286644.
^ Ottesen Mm, Svendsen I (1970). "The subtilisins". Methods Enzymol. 19: 199–215. doi:10.1016/0076-6879(70)19014-8.
^ Markland FS, Smith EL (1971). "Subtilisins: primary structure, chemical and physical properties". In Boyer PD. The Enzymes 3 (3rd ed.). New York: Academic Press. pp. 561–608.
^ Philipp M, Bender ML (1983). "Kinetics of subtilisin and thiolsubtilisin". Mol. Cell. Biochem. 51 (1): 5–32. doi:10.1007/bf00215583. PMID 6343835.
^ Nedkov P, Oberthür W, Braunitzer G (April 1985). "Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus". Biol. Chem. Hoppe-Seyler 366 (4): 421–30. doi:10.1515/bchm3.1985.366.1.421. PMID 3927935.
^ Ikemura H, Takagi H, Inouye M (June 1987). "Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli". J. Biol. Chem. 262 (16): 7859–64. PMID 3108260.
^ Polgár L (1987). "Structure and function of serine proteases". Hydrolytic enzymes. Amsterdam: Elsevier. ISBN 0-444-80886-8.
^ Spar Washing Detergent contents http://www.5010358128009.detergent-info.com/
^ Chaplin, Martin (20 December 2004). "Applications of proteases in the food industry". London South Bank University. Archived from the original on 2010-03-14. Retrieved 3 March 2015.
^ "Callex® Ointment". Archived from the original on 2008-02-03. Retrieved 3 March 2015.

UpToDate Contents

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1. 薬剤の効きづらい高コレステロール血症の治療 treatment of drug resistant hypercholesterolemia
2. LDLコレステロール代謝の主要な障害 primary disorders of ldl cholesterol metabolism
3. 心血管疾患と同等のリスクと確立した危険因子の概要 overview of the risk equivalents and established risk factors for cardiovascular disease
4. 心血管疾患の危険因子の概要 overview of the possible risk factors for cardiovascular disease

English Journal

PCSK9 SNP rs11591147 is associated with low cholesterol levels but not with cognitive performance or noncardiovascular clinical events in an elderly population.

Postmus I, Trompet S, de Craen AJ, Buckley BM, Ford I, Stott DJ, Sattar N, Slagboom PE, Westendorp RG, Jukema JW.SourceDepartments of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands.
Journal of lipid research.J Lipid Res.2013 Feb;54(2):561-6. doi: 10.1194/jlr.P033969.
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a protein involved in LDL-cholesterol metabolism. The single-nucleotide polymorphism (SNP) rs11591147 has been associated with lower LDL-cholesterol and a lower risk of coronary heart disease. Because PCSK9 has high affinity to the LDL re
PMID 23300213

Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets.

Cariou B, Langhi C, Le Bras M, Bortolotti M, Lê KA, Theytaz F, Le May C, Guyomarc H-Delasalle B, Zaïr Y, Kreis R, Boesch C, Krempf M, Tappy L, Costet P.AbstractABSTRACT: BACKGROUND: PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters.
Nutrition & metabolism.Nutr Metab (Lond).2013 Jan 8;10(1):4. [Epub ahead of print]
ABSTRACT: BACKGROUND: PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and
PMID 23298392

Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant.

Al-Mashhadi RH, Sørensen CB, Kragh PM, Christoffersen C, Mortensen MB, Tolbod LP, Thim T, Du Y, Li J, Liu Y, Moldt B, Schmidt M, Vajta G, Larsen T, Purup S, Bolund L, Nielsen LB, Callesen H, Falk E, Mikkelsen JG, Bentzon JF.SourceDepartment of Cardiology, Aarhus University Hospital, and Department of Clinical Medicine, Aarhus University, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Denmark.
Science translational medicine.Sci Transl Med.2013 Jan 2;5(166):166ra1. doi: 10.1126/scitranslmed.3004853.
Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations,
PMID 23283366

Japanese Journal

Comparison of effects of bezafibrate and fenofibrate on circulating proprotein convertase subtilisin/kexin type 9 and adipocytokine levels in dyslipidemic subjects with impaired glucose tolerance or type 2 diabetes mellitus: Results from a crossover study

Noguchi Tohru,Kobayashi Junji,Yagi Kunimasa,Nohara Atsushi,Yamaaki Naoto,Sugihara Masako,Ito Naoko,Oka Rie,Kawashiri Masaaki,Tada Hayato,Takata Mutsuko,Inazu Akihiro,Yamagishi Masakazu,Mabuchi Hiroshi
Atherosclerosis xx(xx), xxxx-xxxx, 2011
… Background: Bezafibrate and fenofibrate show different binding properties against peroxisome proliferator-activated receptor subtypes, which could cause different clinical effects on circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and on various metabolic markers. …
NAID 120003141452

N-terminal deletion of Tk1689, a subtilisin-like serine protease from Thermococcus kodakaraensis, copes with its cytotoxicity in Escherichia coli(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

Rasool Nouman,Rashid Naeem,Iftikhar Saima,Akhtar Muhammad
Journal of bioscience and bioengineering 110(4), 381-385, 2010-10
… Tk1689, a subtilisin-like serine protease from Thermococcus kodakaraensis, was found to be toxic to the host cells when produced in the pro-protein form (Pro-Tk1689) in Eschericia coli. …
NAID 110007880132

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リンク元

「スブチリシン」

Subtilisin BPN'
	Crystal structure of subtilisin.
Identifiers
Symbol	apr
Entrez	5712479
PDB	1st2 More structures
UniProt	P00782
Other data
EC number	3.4.21.62

Search
PMC	articles
PubMed	articles
NCBI	proteins

subtilisin
Identifiers
EC number	3.4.21.62
CAS number	9014-01-1
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / EGO
Search
PMC	articles
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NCBI	proteins

　　[★]

英: subtilisin
同: ズブチリシン、ズブチロペプチダーゼ subtilopeptidase

[pmid3286644-1] PDB 1st2; Bott R, Ultsch M, Kossiakoff A, Graycar T, Katz B, Power S (June 1988). "The three-dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8 A and an analysis of the structural consequences of peroxide inactivation". J. Biol. Chem. 263 (16): 7895–906. PMID 3286644.

[2] Ottesen Mm, Svendsen I (1970). "The subtilisins". Methods Enzymol. 19: 199–215. doi:10.1016/0076-6879(70)19014-8.

[3] Markland FS, Smith EL (1971). "Subtilisins: primary structure, chemical and physical properties". In Boyer PD. The Enzymes 3 (3rd ed.). New York: Academic Press. pp. 561–608.

[pmid6343835-4] Philipp M, Bender ML (1983). "Kinetics of subtilisin and thiolsubtilisin". Mol. Cell. Biochem. 51 (1): 5–32. doi:10.1007/bf00215583. PMID 6343835.

[pmid3927935-5] Nedkov P, Oberthür W, Braunitzer G (April 1985). "Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus". Biol. Chem. Hoppe-Seyler 366 (4): 421–30. doi:10.1515/bchm3.1985.366.1.421. PMID 3927935.

[pmid3108260-6] Ikemura H, Takagi H, Inouye M (June 1987). "Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli". J. Biol. Chem. 262 (16): 7859–64. PMID 3108260.

[isbn0-444-80886-8-7] Polgár L (1987). "Structure and function of serine proteases". Hydrolytic enzymes. Amsterdam: Elsevier. ISBN 0-444-80886-8.

[8] Spar Washing Detergent contents http://www.5010358128009.detergent-info.com/

[9] Chaplin, Martin (20 December 2004). "Applications of proteases in the food industry". London South Bank University. Archived from the original on 2010-03-14. Retrieved 3 March 2015.

[10] "Callex® Ointment". Archived from the original on 2008-02-03. Retrieved 3 March 2015.

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subtilisin