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A catalytic triad is a set of three coordinated
amino acid Amino acids are organic compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compounds are known. The study of the properties, reactions, a ...

amino acid
s that can be found in the
active site Active may refer to: Music * ''Active'' (album), a 1992 album by Casiopea * Active Records Active Records was a record label, record sublabel of RCA Records founded in 1980. The label focused mainly on heavy metal music. The label was disso ...

active site
of some
enzymes Enzymes () are protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958 ...
. Catalytic triads are most commonly found in
hydrolase Hydrolase is a class of enzyme that commonly perform as biochemical catalysts that use water to break a chemical bond, which typically results in dividing a larger molecule into smaller molecules. Some common examples of hydrolase enzymes are este ...
and
transferase '' complexed with α-amanitin (in red). Despite the use of the term "polymerase," RNA polymerases are classified as a form of nucleotidyl transferase. A transferase is any one of a class of enzymes that enact the transfer of specific functional ...
enzymes (e.g.
protease A protease (also called a peptidase or proteinase) is an enzyme Enzymes () are s that act as s (biocatalysts). Catalysts accelerate . The molecules upon which enzymes may act are called , and the enzyme converts the substrates into differe ...

protease
s,
amidase In enzymology Enzymes () are protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery ...
s,
esterase An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water (molecule), water called hydrolysis. A wide range of different esterases exist that differ in their Substrate (biochemistry), subst ...
s, acylases,
lipase A lipase (, ) is any enzyme Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enz ...
s and β-lactamases). An
Acid An acid is a or capable of donating a (hydrogen ion H+) (a ), or, alternatively, capable of forming a with an (a ). The first category of acids are the proton donors, or s. In the special case of , proton donors form the H3O+ and are ...
-
Base Base or BASE may refer to: Brands and enterprises *Base (mobile telephony provider) Base (stylized as BASE) is the third largest of Belgium Belgium ( nl, België ; french: Belgique ; german: Belgien ), officially the Kingdom of Belgium, ...
-
Nucleophile In chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, properties, behavior and the changes they undergo during a ...

Nucleophile
triad is a common motif for generating a nucleophilic residue for
covalent catalysis Enzyme catalysis is the increase in the rate of a process A process is a series or set of activities that interact to produce a result; it may occur once-only or be recurrent or periodic. Things called a process include: Business and manage ...

covalent catalysis
. The form a charge-relay network to polarise and activate the nucleophile, which attacks the
substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached **Substrate (locomotion), the surface over which an organism loco ...
, forming a
covalent A covalent bond is a chemical bond A chemical bond is a lasting attraction between atom An atom is the smallest unit of ordinary matter In classical physics and general chemistry, matter is any substance that has mass and take ...

covalent
intermediate which is then
hydrolysed Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution Substitution may refer to: Arts and media *Chord substitution, in music, swapping one chord fo ...
to release the
product Product may refer to: Business * Product (business) In marketing, a product is an object or system made available for consumer use; it is anything that can be offered to a Market (economics), market to satisfy the desire or need of a customer ...
and regenerate free enzyme. The nucleophile is most commonly a
serine Serine (symbol Ser or S) is an α-amino acid Amino acids are organic compound , CH4; is among the simplest organic compounds. In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen chemical bond, ...

serine
or
cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the chemical formula, formula HOOC-CH-(NH2)-CH2-SH. The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. The thiol is suscepti ...

cysteine
amino acid, but occasionally
threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated ...

threonine
or even
selenocysteine Selenocysteine (symbol Sec or U, in older publications also as Se-Cys) is the 21st proteinogenic amino acid Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation (biology), translation. ...

selenocysteine
. The
3D structure 3-D or 3D or 3d may refer to: Science, technology, and mathematics Relating to three-dimensionality * Three-dimensional space ** 3D computer graphics, computer graphics that use a three-dimensional representation of geometric data ** 3D film, a ...
of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence (
primary structure Biomolecular structure is the intricate folded, three-dimensional shape that is formed by a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon ...

primary structure
). As well as
divergent evolution Divergent evolution or divergent selection is the accumulation of differences between closely related populations within a species, leading to speciation Speciation is the evolution Evolution is change in the Heredity, heritable Phenoty ...
of function (and even the triad's nucleophile), catalytic triads show some of the best examples of
convergent evolution Convergent evolution is the independent evolution Evolution is change in the heritable Heredity, also called inheritance or biological inheritance, is the passing on of Phenotypic trait, traits from parents to their offspring; eit ...
. Chemical constraints on catalysis have led to the same catalytic solution independently evolving in at least 23 separate superfamilies. Their
mechanism of action In pharmacology Pharmacology is a branch of , and concerned with or action, where a drug may be defined as any artificial, natural, or endogenous (from within the body) molecule which exerts a biochemical or physiological effect on the ...

mechanism of action
is consequently one of the best studied in
biochemistry Biochemistry or biological chemistry, is the study of chemical process In a scientific Science () is a systematic enterprise that Scientific method, builds and organizes knowledge in the form of Testability, testable explanations and pr ...

biochemistry
.


History

The enzymes
trypsin Trypsin () is a serine protease Serine proteases (or serine endopeptidases) are s that cleave s in s. serves as the at the (enzyme's) . They are found ubiquitously in both and . Serine proteases fall into two broad categories based on ...
and
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 Digestive may refer to: Biology *Digestion Dige ...

chymotrypsin
were first purified in the 1930s. A serine in each of trypsin and chymotrypsin was identified as the catalytic nucleophile (by
diisopropyl fluorophosphate Diisopropyl fluorophosphate (DFP) or Isoflurophate is an oily, colorless liquid with the chemical formula C6H14FO3P. It is used in medicine and as an organophosphorus compounds, organophosphorus insecticide. It is stable, but undergoes hydrolysis ...
modification) in the 1950s. The structure of chymotrypsin was solved by
X-ray crystallography X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract into many specific directions. By measurin ...
in the 1960s, showing the orientation of the catalytic triad in the active site. Other proteases were sequenced and aligned to reveal a family of related proteases, now called the S1 family. Simultaneously, the structures of the evolutionarily unrelated
papain Papain, also known as papaya proteinase I, is a cysteine protease Cysteine proteases, also known as thiol proteases, are s that degrade s. These s share a common that involves a in a or dyad. Discovered by Gopal Chunder Roy in 1873, the f ...

papain
and
subtilisin Subtilisin is a protease A protease (also called a peptidase or proteinase) is an enzyme Enzymes () are s that act as s (biocatalysts). Catalysts accelerate . The molecules upon which enzymes may act are called , and the enzyme converts ...
proteases were found to contain analogous triads. The 'charge-relay' mechanism for the activation of the nucleophile by the other triad members was proposed in the late 1960s. As more protease structures were solved by
X-ray crystallography X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract into many specific directions. By measurin ...
in the 1970s and 80s, homologous (such as
TEV protease TEV protease (, ''Tobacco Etch Virus nuclear-inclusion-a endopeptidase'') is a highly sequence-specific cysteine protease from Tobacco etch virus, Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases. Due to its h ...
) and analogous (such as papain) triads were found. The
MEROPS MEROPS is an online databaseAn online database is a database In computing Computing is any goal-oriented activity requiring, benefiting from, or creating computing machinery. It includes the study and experimentation of algorithmic processes ...
classification system in the 1990s and 2000s began classing proteases into structurally related enzyme superfamilies and so acts as a database of the convergent evolution of triads in over 20 superfamilies. Understanding how chemical constraints on evolution led to the convergence of so many enzyme families on the same triad
geometries This is a list of geometry Geometry (from the grc, γεωμετρία; ' "earth", ' "measurement") is, with , one of the oldest branches of . It is concerned with properties of space that are related with distance, shape, size, and relat ...

geometries
has developed in the 2010s. Since their initial discovery, there have been increasingly detailed investigations of their exact catalytic mechanism. Of particular contention in the 1990s and 2000s was whether low-barrier hydrogen bonding contributed to catalysis, or whether ordinary
hydrogen bonding A hydrogen bond (or H-bond) is a primarily Electrostatics, electrostatic force of attraction between a hydrogen Hydrogen is the chemical element Image:Simple Periodic Table Chart-blocks.svg, 400px, Periodic table, The periodic table of ...
is sufficient to explain the mechanism. The massive body of work on the charge-relay, covalent catalysis used by catalytic triads has led to the mechanism being the best characterised in all of biochemistry.


Function

Enzymes that contain a catalytic triad use it for one of two reaction types: either to
split Split(s) or The Split may refer to: Places * Split, Croatia, the largest coastal city in Croatia * Split Island, Canada, an island in the Hudson Bay * Split Island, Falkland Islands * Split Island, Fiji, better known as Hạfliua Arts, entertain ...

split
a substrate (
hydrolase Hydrolase is a class of enzyme that commonly perform as biochemical catalysts that use water to break a chemical bond, which typically results in dividing a larger molecule into smaller molecules. Some common examples of hydrolase enzymes are este ...
s) or to transfer one portion of a substrate over to a second substrate (
transferase '' complexed with α-amanitin (in red). Despite the use of the term "polymerase," RNA polymerases are classified as a form of nucleotidyl transferase. A transferase is any one of a class of enzymes that enact the transfer of specific functional ...
s). Triads are an inter-dependent set of residues in the
active site Active may refer to: Music * ''Active'' (album), a 1992 album by Casiopea * Active Records Active Records was a record label, record sublabel of RCA Records founded in 1980. The label focused mainly on heavy metal music. The label was disso ...

active site
of an enzyme and act in concert with other residues (e.g.
binding site Binding may refer to: Computing * Binding, associating a network socket Network and networking may refer to: Arts, entertainment, and media * Network (1976 film), ''Network'' (1976 film), a 1976 American film * Network (2019 film), ''Network'' ...
and
oxyanion hole An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonation, deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues. Stab ...

oxyanion hole
) to achieve nucleophilic catalysis. These triad residues act together to make the
nucleophile In chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, properties, behavior and the changes they undergo during a ...

nucleophile
member highly
reactive Reactive may refer to: *Generally, capable of having a reaction (disambiguation) *An adjective abbreviation denoting a Bowling ball#Coverstock technology, bowling ball coverstock made of reactive resin *Reactivity (chemistry) *Reactive mind *Reacti ...
, generating a covalent intermediate with the substrate that is then resolved to complete catalysis.


Mechanism

Catalytic triads perform
covalent catalysis Enzyme catalysis is the increase in the rate of a process A process is a series or set of activities that interact to produce a result; it may occur once-only or be recurrent or periodic. Things called a process include: Business and manage ...

covalent catalysis
using a residue as a nucleophile. The reactivity of the nucleophilic residue is increased by the
functional group In organic chemistry, a functional group is a substituent or moiety (chemistry), moiety in a molecule that causes the molecule's characteristic chemical reactions. The same functional group will undergo the same or similar chemical reactions re ...
s of the other triad members. The nucleophile is polarised and oriented by the base, which is itself bound and stabilised by the acid. Catalysis is performed in two stages. First, the activated nucleophile attacks the
carbonyl In organic chemistry Organic chemistry is a branch of chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, pro ...

carbonyl
carbon and forces the carbonyl oxygen to accept an electron, leading to a tetrahedral intermediate. The build-up of negative charge on this intermediate is typically stabilized by an
oxyanion hole An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonation, deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues. Stab ...

oxyanion hole
within the active site. The intermediate then collapses back to a carbonyl, ejecting the first half of the substrate, but leaving the second half still covalently bound to the enzyme as an acyl-enzyme intermediate. The ejection of this first
leaving group In chemistry Chemistry is the scientific Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a familiarity or awareness, of someone or something, such as facts A fact is an occurrence in the ...
is often aided by donation of a proton by the base. The second stage of catalysis is the resolution of the acyl-enzyme intermediate by the attack of a second substrate. If this substrate is water then the result is hydrolysis; if it is an organic molecule then the result is transfer of that molecule onto the first substrate. Attack by this second substrate forms a new tetrahedral intermediate, which resolves by ejecting the enzyme's nucleophile, releasing the second product and regenerating free enzyme.


Identity of triad members


Nucleophile

The side-chain of the nucleophilic residue performs covalent catalysis on the
substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached **Substrate (locomotion), the surface over which an organism loco ...
. The
lone pair In chemistry Chemistry is the scientific Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a familiarity or awareness, of someone or something, such as facts A fact is an occurrence in the ...
of electrons present on the oxygen or sulfur attacks the electropositive
carbonyl In organic chemistry Organic chemistry is a branch of chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, pro ...

carbonyl
carbon. The 20 naturally occurring biological amino acids do not contain any sufficiently nucleophilic functional groups for many difficult catalytic reactions. Embedding the nucleophile in a triad increases its reactivity for efficient catalysis. The most commonly used nucleophiles are the
hydroxyl A hydroxy or hydroxyl group is a functional group with the chemical formula -OH and composed of one oxygen Oxygen is the chemical element Image:Simple Periodic Table Chart-blocks.svg, 400px, Periodic table, The periodic table of the ...

hydroxyl
(OH) of serine and the
thiol A thiol () or thiol derivative is any organosulfur compound Organosulfur compounds are organic compound , CH4; is among the simplest organic compounds. In chemistry Chemistry is the scientific discipline involved with Chemical element, elem ...

thiol
/thiolate ion (SH/S) of cysteine. Alternatively,
threonine protease Threonine proteases are a family of proteolytic enzymes harbouring a threonine (Thr) residue within the active site. The prototype members of this class of enzymes are the catalysis, catalytic subunits of the proteasome, however the acyltransferas ...
s use the of threonine, however due to
steric hindrance Steric effects are nonbonding interactions that influence the shape (conformation Conformation generally means structural arrangement and may refer to: * Conformational isomerism, a form of stereoisomerism in chemistry ** Carbohydrate conformat ...
of the side chain's extra
methyl group A methyl group is an alkyl In organic chemistry Organic chemistry is a branch of chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: t ...

methyl group
such proteases use their ''N''-terminal amide as the base, rather than a separate amino acid. Use of oxygen or sulfur as the nucleophilic atom causes minor differences in catalysis. Compared to
oxygen Oxygen is the chemical element Image:Simple Periodic Table Chart-blocks.svg, 400px, Periodic table, The periodic table of the chemical elements In chemistry, an element is a pure substance consisting only of atoms that all have the same ...

oxygen
,
sulfur Sulfur (in nontechnical British English: sulphur) is a chemical element In chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: th ...

sulfur
's extra
d orbital In atomic theory Atomic theory is the scientific theory A scientific theory is an explanation of an aspect of the natural world and universe that has been repeatedly tested and verified in accordance with the scientific method The ...

d orbital
makes it larger (by 0.4 Å) and softer, allows it to form longer bonds (dC-X and dX-H by 1.3-fold), and gives it a lower p''K''a (by 5 units). Serine is therefore more dependent than cysteine on optimal orientation of the acid-base triad members to reduce its p''K''a in order to achieve concerted
deprotonation Deprotonation (or dehydronation) is the removal (transfer) of a proton A proton is a subatomic particle, symbol or , with a positive electric charge of +1''e'' elementary charge and a mass slightly less than that of a neutron. Protons and neutr ...

deprotonation
with catalysis. The low p''K''a of cysteine works to its disadvantage in the resolution of the first
tetrahedral intermediate A tetrahedral intermediate is a reaction intermediate A reaction intermediate or an intermediate is a molecular entityA molecular entity, or chemical entity, is "any constitutionally or isotopically distinct atom, molecule, ion, ion pair, Radical ...
as unproductive reversal of the original nucleophilic attack is the more favourable breakdown product. The triad base is therefore preferentially oriented to
protonate In chemistry, protonation (or hydronation) is the adding of a proton#Hydrogen ion, proton (or hydron (chemistry), hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is ...
the leaving group amide to ensure that it is ejected to leave the enzyme sulfur covalently bound to the substrate N-terminus. Finally, resolution of the acyl-enzyme (to release the substrate C-terminus) requires serine to be re-protonated whereas cysteine can leave as S. Sterically, the sulfur of cysteine also forms longer bonds and has a bulkier
van der Waals radius The Van der Waals radius, ''r'', of an atom An atom is the smallest unit of ordinary matter In classical physics and general chemistry, matter is any substance that has mass and takes up space by having volume. All everyday objects that ...
and if
mutated In biology, a mutation is an alteration in the base sequence, nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA replication, DNA o ...
to serine can be trapped in unproductive orientations in the active site. Very rarely, the
selenium Selenium is a chemical element upright=1.0, 500px, The chemical elements ordered by link=Periodic table In chemistry Chemistry is the science, scientific study of the properties and behavior of matter. It is a natural science that ...

selenium
atom of the uncommon amino acid
selenocysteine Selenocysteine (symbol Sec or U, in older publications also as Se-Cys) is the 21st proteinogenic amino acid Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation (biology), translation. ...

selenocysteine
is used as a nucleophile. The deprotonated Se state is strongly favoured when in a catalytic triad.


Base

Since no natural amino acids are strongly nucleophilic, the base in a catalytic triad polarises and
deprotonate Deprotonation (or dehydronation) is the removal (transfer) of a proton A proton is a subatomic particle, symbol or , with a positive electric charge of +1''e'' elementary charge and a mass slightly less than that of a neutron. Protons and neutr ...
s the nucleophile to increase its reactivity. Additionally, it
protonate In chemistry, protonation (or hydronation) is the adding of a proton#Hydrogen ion, proton (or hydron (chemistry), hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is ...
s the first
product Product may refer to: Business * Product (business) In marketing, a product is an object or system made available for consumer use; it is anything that can be offered to a Market (economics), market to satisfy the desire or need of a customer ...
to aid leaving group departure. The base is most commonly histidine since its p''K''a allows for effective base catalysis, hydrogen bonding to the acid residue, and deprotonation of the nucleophile residue. β-lactamases such as TEM-1 use a
lysine Lysine (symbol Lys or K) is an α-amino acid Amino acids are organic compound , CH4; is among the simplest organic compounds. In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen chemical bond, ...

lysine
residue as the base. Because lysine's p''K''a is so high (p''K''a=11), a glutamate and several other residues act as the acid to stabilise its deprotonated state during the catalytic cycle. Threonine proteases use their ''N''-terminal amide as the base, since steric crowding by the catalytic threonine's methyl prevents other residues from being close enough.


Acid

The acidic triad member forms a hydrogen bond with the basic residue. This aligns the basic residue by restricting its side-chain rotation, and polarises it by stabilising its positive charge. Two amino acids have acidic
side chains In organic chemistry Organic chemistry is a branch of chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, p ...
at physiological pH (aspartate or glutamate) and so are the most commonly used for this triad member.
Cytomegalovirus ''Cytomegalovirus'' (''CMV'') (from ''cyto-'' 'cell' via Greek#REDIRECT Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country located in Sou ...
protease uses a pair of histidines, one as the base, as usual, and one as the acid. The second histidine is not as effective an acid as the more common aspartate or glutamate, leading to a lower catalytic efficiency. In some enzymes, the acid member of the triad is less necessary and some act only as a dyad. For example,
papain Papain, also known as papaya proteinase I, is a cysteine protease Cysteine proteases, also known as thiol proteases, are s that degrade s. These s share a common that involves a in a or dyad. Discovered by Gopal Chunder Roy in 1873, the f ...

papain
uses
asparagine Asparagine (symbol Asn or N), is an α-amino acid Amino acids are organic compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compound ...

asparagine
as its third triad member which orients the histidine base but does not act as an acid. Similarly,
hepatitis A Hepatitis A is an infectious disease of the liver caused by ''Hepatovirus A'' (HAV); it is a type of viral hepatitis. Many cases have few or no symptoms, especially in the young. The time between infection and symptoms, in those who develop them ...
virus protease contains an ordered water in the position where an acid residue should be.


Examples of triads


Ser-His-Asp

The Serine-Histidine-Aspartate motif is one of the most thoroughly characterised catalytic motifs in biochemistry. The triad is exemplified by
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 Digestive may refer to: Biology *Digestion Dige ...

chymotrypsin
, a model serine protease from the PA superfamily which uses its triad to hydrolyse protein backbones. The aspartate is hydrogen bonded to the histidine, increasing the p''K''a of its imidazole nitrogen from 7 to around 12. This allows the histidine to act as a powerful general base and to activate the serine nucleophile. It also has an
oxyanion hole An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonation, deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues. Stab ...

oxyanion hole
consisting of several backbone amides which stabilises charge build-up on intermediates. The histidine base aids the first leaving group by donating a proton, and also activates the hydrolytic water substrate by abstracting a proton as the remaining OH attacks the acyl-enzyme intermediate. The same triad has also convergently evolved in α/β hydrolases such as some
lipase A lipase (, ) is any enzyme Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enz ...
s and
esterase An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water (molecule), water called hydrolysis. A wide range of different esterases exist that differ in their Substrate (biochemistry), subst ...
s, however
orientation Orientation may refer to: Positioning in physical space * Map orientation, the relationship between directions on a map and compass directions * Orientation (housing), the position of a building with respect to the sun, a concept in building desi ...

orientation
of the triad members is reversed. Additionally, brain acetyl hydrolase (which has the same fold as a small
G-protein G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switch A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A ...

G-protein
) has also been found to have this triad. The equivalent Ser-His-''Glu ''triad is used in
acetylcholinesterase Acetylcholinesterase ( HGNC symbol ACHE; EC 3.1.1.7), also known as AChE or acetylhydrolase, is the primary cholinesterase In biochemistry Biochemistry or biological chemistry, is the study of es within and relating to living s. A su ...

acetylcholinesterase
.


Cys-His-Asp

The second most studied triad is the Cysteine-Histidine-Aspartate motif. Several families of
cysteine protease Cysteine proteases, also known as thiol proteases, are hydrolase Hydrolase is a class of enzyme that commonly perform as biochemical catalysts that use water to break a chemical bond, which typically results in dividing a larger molecule into sma ...
s use this triad set, for example TEV protease and
papain Papain, also known as papaya proteinase I, is a cysteine protease Cysteine proteases, also known as thiol proteases, are s that degrade s. These s share a common that involves a in a or dyad. Discovered by Gopal Chunder Roy in 1873, the f ...

papain
. The triad acts similarly to serine protease triads, with a few notable differences. Due to cysteine's low p''K''a, the importance of the Asp to catalysis varies and several cysteine proteases are effectively Cys-His dyads (e.g.
hepatitis A virus Hepatitis A is an infectious disease of the liver caused by ''Hepatovirus A'' (HAV); it is a type of viral hepatitis. Many cases have few or no symptoms, especially in the young. The time between infection and symptoms, in those who develop them ...
protease), whilst in others the cysteine is already deprotonated before catalysis begins (e.g. papain). This triad is also used by some amidases, such as ''N''-glycanase to hydrolyse non-peptide C-N bonds.


Ser-His-His

The triad of
cytomegalovirus ''Cytomegalovirus'' (''CMV'') (from ''cyto-'' 'cell' via Greek#REDIRECT Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country located in Sou ...
protease uses histidine as both the acid and base triad members. Removing the acid histidine results in only a 10-fold activity loss (compared to >10,000-fold when aspartate is removed from chymotrypsin). This triad has been interpreted as a possible way of generating a less active enzyme to control cleavage rate.


Ser-Glu-Asp

An unusual triad is found in seldolisin proteases. The low p''K''a of the glutamate carboxylate group means that it only acts as a base in the triad at very low pH. The triad is hypothesised to be an
adaptation In , adaptation has three related meanings. Firstly, it is the dynamic evolutionary process that fits s to their environment, enhancing their . Secondly, it is a state reached by the population during that process. Thirdly, it is a or adapti ...

adaptation
to specific environments like
acidic An acid is a molecule A molecule is an electrically Electricity is the set of physical phenomena associated with the presence and motion Image:Leaving Yongsan Station.jpg, 300px, Motion involves a change in position In phys ...
hot spring A hot spring, hydrothermal spring, or geothermal spring is a spring Spring(s) may refer to: Common uses * Spring (season), a season of the year * Spring (device), a mechanical device that stores energy * Spring (hydrology), a natural source of w ...

hot spring
s (e.g. kumamolysin) or cell
lysosome A lysosome () is a membrane-bound organelle found in many animal Cell (biology), cells. They are spherical Vesicle (biology and chemistry), vesicles that contain Hydrolysis, hydrolytic enzymes that can break down many kinds of biomolecules. A ly ...

lysosome
(e.g. tripeptidyl peptidase).


Cys-His-Ser

The
endothelial Endothelium is a single layer of squamous Epithelium () is one of the four basic types of animal Animals (also called Metazoa) are multicellular eukaryotic organisms that form the Kingdom (biology), biological kingdom Animalia. Wit ...
protease vasohibin uses a cysteine as the nucleophile, but a serine to coordinate the histidine base. Despite the serine being a poor acid, it is still effective in orienting the histidine in the catalytic triad. Some homologues alternatively have a threonine instead of serine at the acid location.


Thr-Nter, Ser-Nter and Cys-Nter

Threonine proteases, such as the
proteasome Proteasomes are protein complex A protein complex or multiprotein complex is a group of two or more associated polypeptide chain Peptides (from Greek language Greek (modern , romanized: ''Elliniká'', Ancient Greek, ancient , ''Hellēn ...

proteasome
protease subunit and ornithine acyltransferases use the secondary hydroxyl of threonine in a manner analogous to the use of the serine . However, due to the steric interference of the extra methyl group of threonine, the base member of the triad is the ''N''-terminal amide which polarises an ordered water which, in turn, deprotonates the catalytic hydroxyl to increase its reactivity. Similarly, there exist equivalent 'serine only' and 'cysteine only' configurations such as penicillin acylase G and penicillin acylase V which are evolutionarily related to the proteasome proteases. Again, these use their ''N''-terminal amide as a base.


Ser-''cis''Ser-Lys

This unusual triad occurs only in one superfamily of amidases. In this case, the lysine acts to polarise the middle serine. The middle serine then forms two strong hydrogen bonds to the nucleophilic serine to activate it (one with the side chain hydroxyl and the other with the backbone amide). The middle serine is held in an unusual ''Cis–trans isomerism, cis'' orientation to facilitate precise contacts with the other two triad residues. The triad is further unusual in that the lysine and ''cis''-serine both act as the base in activating the catalytic serine, but the same lysine also performs the role of the acid member as well as making key structural contacts.


Sec-His-Glu

The rare, but naturally occurring amino acid
selenocysteine Selenocysteine (symbol Sec or U, in older publications also as Se-Cys) is the 21st proteinogenic amino acid Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation (biology), translation. ...

selenocysteine
(Sec), can also be found as the nucleophile in some catalytic triads. Selenocysteine is similar to cysteine, but contains a
selenium Selenium is a chemical element upright=1.0, 500px, The chemical elements ordered by link=Periodic table In chemistry Chemistry is the science, scientific study of the properties and behavior of matter. It is a natural science that ...

selenium
atom instead of a sulfur. An example is in the active site of thioredoxin reductase, which uses the selenium for reduction of disulfide in thioredoxin.


Engineered triads

In addition to naturally occurring types of catalytic triads, protein engineering has been used to create enzyme variants with non-native amino acids, or entirely synthetic amino acids. Catalytic triads have also been inserted into otherwise non-catalytic proteins, or protein mimics. Subtilisin (a serine protease) has had its oxygen nucleophile replaced with each of sulfur,
selenium Selenium is a chemical element upright=1.0, 500px, The chemical elements ordered by link=Periodic table In chemistry Chemistry is the science, scientific study of the properties and behavior of matter. It is a natural science that ...

selenium
, or tellurium. Cysteine and selenocysteine were inserted by Site-directed mutagenesis, mutagenesis, whereas the non-natural amino acid, tellurocysteine, was inserted using Auxotrophy, auxotrophic cells fed with synthetic tellurocysteine. These elements are all in the 16th Group (periodic table), periodic table column (chalcogens), so have similar properties. In each case, changing the nucleophile reduced the enzyme's protease activity, but increased a different activity. A sulfur nucleophile improved the enzymes
transferase '' complexed with α-amanitin (in red). Despite the use of the term "polymerase," RNA polymerases are classified as a form of nucleotidyl transferase. A transferase is any one of a class of enzymes that enact the transfer of specific functional ...
activity (sometimes called subtiligase). Selenium and tellurium nucleophiles converted the enzyme into an oxidoreductase. When the nucleophile of TEV protease was converted from cysteine to serine, it protease activity was strongly reduced, but was able to be restored by directed evolution. Non-catalytic proteins have been used as scaffolds, having catalytic triads inserted into them which were then improved by directed evolution. The Ser-His-Asp triad has been inserted into an antibody, as well as a range of other proteins. Similarly, catalytic triad mimics have been created in Small molecule, small organic molecules like diaryl diselenide, and displayed on larger polymers like Merrifield resins, and Self-assembling peptide, self-assembling short peptide nanostructures.


Divergent evolution

The sophistication of the active site network causes residues involved in catalysis (and residues in contact with these) to be highly evolutionarily conserved. However, there are examples of divergent evolution in catalytic triads, both in the reaction catalysed, and the residues used in catalysis. The triad remains the core of the active site, but it is evolutionarily adaptation, adapted to serve different functions. Some proteins, called pseudoenzymes, have non-catalytic functions (e.g. regulation by inhibitory binding) and have accumulated mutations that inactivate their catalytic triad.


Reaction changes

Catalytic triads perform
covalent catalysis Enzyme catalysis is the increase in the rate of a process A process is a series or set of activities that interact to produce a result; it may occur once-only or be recurrent or periodic. Things called a process include: Business and manage ...

covalent catalysis
via an acyl-enzyme intermediate. If this intermediate is resolved by water, the result is hydrolysis of the substrate. However, if the intermediate is resolved by attack by a second substrate, then the enzyme acts as a
transferase '' complexed with α-amanitin (in red). Despite the use of the term "polymerase," RNA polymerases are classified as a form of nucleotidyl transferase. A transferase is any one of a class of enzymes that enact the transfer of specific functional ...
. For example, attack by an acyl group results in an acyltransferase reaction. Several families of transferase enzymes have evolved from hydrolases by adaptation to exclude water and favour attack of a second substrate. In different members of the α/β-hydrolase superfamily, the Ser-His-Asp triad is tuned by surrounding residues to perform at least 17 different reactions. Some of these reactions are also achieved with mechanisms that have altered formation, or resolution of the acyl-enzyme intermediate, or that don't proceed via an acyl-enzyme intermediate. Additionally, an alternative transferase mechanism has been evolved by amidophosphoribosyltransferases, which has two active sites. In the first active site, a cysteine triad hydrolyses a glutamine substrate to release free ammonia. The ammonia then diffuses though an enzyme tunnel, internal tunnel in the enzyme to the second active site, where it is transferred to a second substrate.


Nucleophile changes

Divergent evolution of active site residues is slow, due to strong chemical constraints. Nevertheless, some protease Protein superfamily, superfamilies have evolved from one nucleophile to another. This can be inferred when a superfamily (with the same protein structure, fold) contains protein family, families that use different nucleophiles. Such nucleophile switches have occurred several times during evolutionary history, however the mechanisms by which this happen are still unclear. Within protease superfamilies that contain a mixture of nucleophiles (e.g. the PA clan), families are designated by their catalytic nucleophile (C=cysteine proteases, S=serine proteases).


Pseudoenzymes

A further subclass of catalytic triad variants are pseudoenzymes, which have triad mutations that make them catalytically inactive, but able to function as binding or structural proteins. For example, the heparin-binding protein Azurocidin 1, Azurocidin is a member of the PA clan, but with a glycine in place of the nucleophile and a serine in place of the histidine. Similarly, RHBDF1 is a homolog of the S54 family rhomboid proteases with an alanine in the place of the nucleophilic serine. In some cases, pseudoenzymes may still have an intact catalytic triad but mutations in the rest of the protein remove catalytic activity. The CA clan contains catalytically inactive members with mutated triads (calpamodulin has lysine in place of its cysteine nucleophile) and with intact triads but inactivating mutations elsewhere (rat testin retains a Cys-His-Asn triad).


Convergent evolution

The enzymology of proteases provides some of the clearest known examples of convergent evolution. The same geometric arrangement of triad residues occurs in over 20 separate enzyme Superfamily (proteins), superfamilies. Each of these superfamilies is the result of convergent evolution for the same triad arrangement within a different Protein tertiary structure, structural fold. This is because there are limited productive ways to arrange three triad residues, the enzyme backbone and the substrate. These examples reflect the intrinsic chemical and physical constraints on enzymes, leading evolution to repeatedly and independently converge on equivalent solutions.


Cysteine and serine hydrolases

The same triad geometries been converged upon by serine proteases such as 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 Digestive may refer to: Biology *Digestion Dige ...

chymotrypsin
and
subtilisin Subtilisin is a protease A protease (also called a peptidase or proteinase) is an enzyme Enzymes () are s that act as s (biocatalysts). Catalysts accelerate . The molecules upon which enzymes may act are called , and the enzyme converts ...
superfamilies. Similar convergent evolution has occurred with cysteine proteases such as viral C3 protease and
papain Papain, also known as papaya proteinase I, is a cysteine protease Cysteine proteases, also known as thiol proteases, are s that degrade s. These s share a common that involves a in a or dyad. Discovered by Gopal Chunder Roy in 1873, the f ...

papain
superfamilies. These triads have converged to almost the same arrangement due to the mechanistic similarities in cysteine and serine proteolysis mechanisms. Families of cysteine proteases Families of serine proteases


Threonine proteases

Threonine proteases use the amino acid threonine as their catalytic nucleophile. Unlike cysteine and serine, threonine is a secondary hydroxyl (i.e. has a methyl group). This methyl group greatly restricts the possible orientations of triad and substrate as the methyl clashes with either the enzyme backbone or histidine base. When the nucleophile of a serine protease was mutated to threonine, the methyl occupied a mixture of positions, most of which prevented substrate binding. Consequently, the catalytic residue of a threonine protease is located at its ''N''-terminus. Two evolutionarily independent enzyme superfamilies with different protein folds are known to use the ''N''-terminal residue as a nucleophile: Superfamily PB (proteasomes using the Ntn fold) and Superfamily PE (acetyltransferases using the DOM fold) This commonality of
active site Active may refer to: Music * ''Active'' (album), a 1992 album by Casiopea * Active Records Active Records was a record label, record sublabel of RCA Records founded in 1980. The label focused mainly on heavy metal music. The label was disso ...

active site
structure in completely different protein folds indicates that the active site evolved convergently in those superfamilies. Families of threonine proteases


See also

*Active site *Convergent evolution *Divergent evolution *Enzyme catalysis *Enzyme superfamily *Functional groups *PA clan *Protease *Proteolysis


References


Notes


Citations

{{Use British English, date=August 2015 Molecular biology Catalysis Evolution