Subtilisin
Encyclopedia
Subtilisin is a non-specific protease
(a protein-digesting
enzyme
) initially obtained from Bacillus subtilis
.
Subtilisins belong to subtilase
s, a group of serine protease
s that initiate the nucleophilic attack on the peptide
(amide) bond through a serine residue
at the active site
. They are physically and chemically well-characterized enzymes. Subtilisins typically have molecular weights of about 20,000 to 45,000 dalton. They can be obtained from soil bacteria, for example, Bacillus amyloliquefaciens
. Subtilisins are secreted in large amounts from many Bacillus species.
Subtilisins are widely used in commercial products, for example, in laundry and dishwashing detergents, cosmetics, food processing, skin care ointments, contact lens cleaners, and for research purposes in synthetic organic chemistry
.
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 the classic example of convergent evolution
.
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.
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 bond
s with its partially negative oxygen atom. This is possible due to the nature of the charge-relay site of subtilisin.
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
(a protein-digesting
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
) initially obtained from Bacillus subtilis
Bacillus subtilis
Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate...
.
Subtilisins belong to subtilase
Subtilase
Subtilases are a family of subtilisin-like serine proteases. They appear to have independently and convergently evolved an Asp/Ser/His catalytic triad, like in the trypsin serine proteases...
s, a group of serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...
s that initiate the nucleophilic attack on the peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
(amide) bond through a serine residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....
at the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
. They are physically and chemically well-characterized enzymes. Subtilisins typically have molecular weights of about 20,000 to 45,000 dalton. They can be obtained from soil bacteria, for example, Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens is a species of bacteria that is the source of the BamH1 restriction enzyme. It also synthesizes a natural antibiotic protein barnase, a widely studied ribonuclease that forms a famously tight complex with its intracellular inhibitor barstar.-Discovery and name:B...
. Subtilisins are secreted in large amounts from many Bacillus species.
Subtilisins are widely used in commercial products, for example, in laundry and dishwashing detergents, cosmetics, food processing, skin care ointments, contact lens cleaners, and for research purposes in synthetic organic chemistry
Organic chemistry
Organic chemistry is a subdiscipline within chemistry involving the scientific study of the structure, properties, composition, reactions, and preparation of carbon-based compounds, hydrocarbons, and their derivatives...
.
The structure of subtilisin has been determined by X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
. It is a 275-residue globular protein
Globular protein
Globular proteins, or spheroproteins are one of the two main protein classes, comprising "globe"-like proteins that are more or less soluble in aqueous solutions...
with several alpha-helices, and a large beta-sheet. It is structurally unrelated to the chymotrypsin
Chymotrypsin
Chymotrypsin is a digestive enzyme that can perform proteolysis. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond is a tyrosine, tryptophan, or phenylalanine. These amino acids contain an aromatic ring in their sidechain that fits into a...
-clan of serine proteases, but uses the same type of catalytic triad
Catalytic triad
A catalytic triad refers to the three amino acid residues found inside the active site of certain protease enzymes: serine , aspartate , and histidine . They work together to break peptide bonds on polypeptides. In general terms, catalytic triad can refer to any set of three residues that function...
in the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
. This makes it the classic example of convergent evolution
Convergent evolution
Convergent evolution describes the acquisition of the same biological trait in unrelated lineages.The wing is a classic example of convergent evolution in action. Although their last common ancestor did not have wings, both birds and bats do, and are capable of powered flight. The wings are...
.
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.
Charge-relay Site of Subtilisin
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 imidazoleImidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
ring. This is possible because Asp is negatively charged at physiological pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
. 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
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
to form the active site.
To summarize the interactions described above, Ser-221 acts as a nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
and cleaves peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...
s with its partially negative oxygen atom. This is possible due to the nature of the charge-relay site of subtilisin.