Subtilisin is a

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

(a protein-digesting

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

) initially obtained from ''

Bacillus subtilis ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...

''. Subtilisins belong to

subtilase Subtilases are a protein family, family of subtilisin-like serine proteases. They appear to have independently and convergently evolved an Aspartate, Asp/Serine, Ser/Histidine, His catalytic triad, like in the trypsin, trypsin serine proteases. Th ...

s, a group of

serine protease Serine proteases (or serine endopeptidases) are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serin ...

s that – like all serine proteases – initiate the

nucleophilic In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they a ...

attack on the

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

(amide) bond through a serine residue at the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

. Subtilisins typically have molecular weights 27kDa. They can be obtained from certain types of

soil Soil, also commonly referred to as earth, is a mixture of organic matter, minerals, gases, water, and organisms that together support the life of plants and soil organisms. Some scientific definitions distinguish dirt from ''soil'' by re ...

bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...

, 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 In biology, a clade (), also known as a Monophyly, monophyletic group or natural group, is a group of organisms that is composed of a common ancestor and all of its descendants. Clades are the fundamental unit of cladistics, a modern approach t ...

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). 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 X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

. The mature form is a 275-residue

globular protein In biochemistry, globular proteins or spheroproteins are spherical ("globe-like") proteins and are one of the common protein types (the others being fibrous, disordered and membrane proteins). Globular proteins are somewhat water-soluble (form ...

with several

alpha-helices An alpha helix (or α-helix) is a sequence of amino acids in a protein that are twisted into a coil (a helix). The alpha helix is the most common structural arrangement in the secondary structure of proteins. It is also the most extreme type of l ...

, and a large

beta-sheet The beta sheet (β-sheet, also β-pleated sheet) is a common structural motif, motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone chain, backbon ...

. The N-terminal contains an I9 propeptide domain () that assists the folding of subtilisin. Proteolytic removal of the domain activates the enzyme. It is structurally unrelated to the

chymotrypsin Chymotrypsin (, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenu ...

-clan of serine proteases, but uses the same type of

catalytic triad A catalytic triad is a set of three coordinated amino acid residues that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, aminoac ...

in the

. This makes it a classic example of

convergent evolution Convergent evolution is the independent evolution of similar features in species of different periods or epochs in time. Convergent evolution creates analogous structures that have similar form or function but were not present in the last comm ...

Mechanism of catalysis

The active site features a charge-relay network involving Asp-32, His-64, and active site Ser-221 arranged in a

. 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 Imidazole (ImH) is an organic compound with the formula . It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. It can be classified as a heterocycle, specifically as a diazole. Many natural products, ...

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 In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...

and cleaves

peptide bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...

s 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 A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms. Mo ...

, 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 and dishwashing detergents,

cosmetics Cosmetics are substances that are intended for application to the body for cleansing, beautifying, promoting attractiveness, or altering appearance. They are mixtures of chemical compounds derived from either Natural product, natural source ...

food processing Food processing is the transformation of agricultural products into food, or of one form of food into other forms. Food processing takes many forms, from grinding grain into raw flour, home cooking, and complex industrial methods used in the mak ...

, skin care products,

contact lens Contact lenses, or simply contacts, are thin lenses placed directly on the surface of the eyes. Contact lenses are ocular prosthetic devices used by over 150 million people worldwide, and they can be worn to correct vision or for cosmetic ...

cleaners, and for research in

synthetic organic chemistry Organic chemistry is a subdiscipline within chemistry involving the scientific study of the structure, properties, and reactions of organic compounds and organic materials, i.e., matter in its various forms that contain carbon atoms.Clayden, J ...

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 The National Institute for Occupational Safety and Health (NIOSH, ) is the List of United States federal agencies, United States federal agency responsible for conducting research and making recommendations for the prevention of work-related occ ...

(NIOSH) has set a

recommended exposure limit A recommended exposure limit (REL) is an occupational exposure limit that has been recommended by the United States National Institute for Occupational Safety and Health. The REL is a level that NIOSH believes would be protective of worker safety ...

(REL) of 60 ng/m³ over a 60-minute period. 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

References

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