Catalytic triad

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aspartate (acid), histidine (base) and cysteine (nucleophile). The substrate (black) is bound by the binding site to orient it next to the triad. (PDB: 1LVM
​)

A catalytic triad is a set of three coordinated

hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence (primary structure).[3]

As well as

mechanism of action is consequently one of the best studied in biochemistry.[4][5]

History

The enzymes

structurally related enzyme superfamilies and so acts as a database of the convergent evolution of triads in over 20 superfamilies.[16][17] Understanding how chemical constraints on evolution led to the convergence of so many enzyme families on the same triad geometries has developed in the 2010s.[2]

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

hydrogen bonding is sufficient to explain the mechanism.[21][22] 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.[4][5][21]

Function

Enzymes that contain a catalytic triad use it for one of two reaction types: either to

nucleophilic catalysis. These triad residues act together to make the nucleophile member highly reactive, generating a covalent intermediate with the substrate that is then resolved to complete catalysis.[citation needed
]

Mechanism

Catalytic triads perform

covalent catalysis using a residue as a nucleophile. The reactivity of the nucleophilic residue is increased by the functional groups of the other triad members. The nucleophile is polarised and oriented by the base, which is itself bound and stabilised by the acid.[citation needed
]

Catalysis is performed in two stages. First, the activated nucleophile attacks the

acyl-enzyme intermediate. Although general-acid catalysis for breakdown of the First and Second tetrahedral intermediate may occur by the path shown in the diagram, evidence supporting this mechanism with chymotrypsin[23] has been controverted.[24]

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.[25]

carbonyl substrate (red) by a second substrate (blue). First, the enzyme's nucleophile (X) attacks the carbonyl to form a covalently linked acyl-enzyme intermediate. This intermediate is then attacked by the second substrate's nucleophile (X'). If the second nucleophile is the hydroxyl of water, the result is hydrolysis, otherwise the result is group transfer
of X'.

Identity of triad members

pKa of the nucleophilic residue which then attacks the substrate. An oxyanion hole of positively charged usually backbone amides (occasionally side-chains) stabilise charge build-up on the substrate transition state
.

Nucleophile

The side-chain of the nucleophilic residue performs covalent catalysis on the

N-terminal amide as the base, rather than a separate amino acid.[1][26]

Use of oxygen or sulfur as the nucleophilic atom causes minor differences in catalysis. Compared to

mutated to serine can be trapped in unproductive orientations in the active site.[27]

Very rarely, the selenium atom of the uncommon amino acid selenocysteine is used as a nucleophile.[29] The deprotonated Se− state is strongly favoured when in a catalytic triad.[29]

Base

Since no natural amino acids are strongly nucleophilic, the base in a catalytic triad

protonates the first product to aid leaving group departure.[citation needed
]

The base is most commonly histidine since its pKa allows for effective base catalysis, hydrogen bonding to the acid residue, and deprotonation of the nucleophile residue.

TEM-1 use a lysine residue as the base. Because lysine's pKa is so high (pKa=11), a glutamate and several other residues act as the acid to stabilise its deprotonated state during the catalytic cycle.[30][31] 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.[32][33]

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.[3] Two amino acids have acidic side chains at physiological pH (aspartate or glutamate) and so are the most commonly used for this triad member.[3] Cytomegalovirus protease[b] uses a pair of histidines, one as the base, as usual, and one as the acid.[1] 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[c] uses asparagine as its third triad member which orients the histidine base but does not act as an acid. Similarly, hepatitis A virus protease[d] contains an ordered water in the position where an acid residue should be.[citation needed]

Examples of triads

penicillin acylase and 2 by beta-lactamase
).

Ser-His-Asp

The Serine-Histidine-Aspartate motif is one of the most thoroughly characterised catalytic motifs in biochemistry.

PA superfamily which uses its triad to hydrolyse protein backbones. The aspartate is hydrogen bonded to the histidine, increasing the pKa 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 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.[citation needed
]

The same triad has also convergently evolved in

G-protein) has also been found to have this triad. The equivalent Ser-His-Glu triad is used in acetylcholinesterase.[citation needed
]

Cys-His-Asp

The second most studied triad is the Cysteine-Histidine-Aspartate motif.[2] Several families of cysteine proteases use this triad set, for example TEV protease[a] and papain.[c] The triad acts similarly to serine protease triads, with a few notable differences. Due to cysteine's low pKa, the importance of the Asp to catalysis varies and several cysteine proteases are effectively Cys-His dyads (e.g. hepatitis A virus protease), whilst in others the cysteine is already deprotonated before catalysis begins (e.g. papain).[36] This triad is also used by some amidases, such as N-glycanase to hydrolyse non-peptide C-N bonds.[37]

Ser-His-His

The triad of cytomegalovirus protease[b] 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.[26]

Ser-Glu-Asp

An unusual triad is found in

acidic hot springs (e.g. kumamolysin) or cell lysosome (e.g. tripeptidyl peptidase).[26]

Cys-His-Ser

The endothelial protease vasohibin[g] uses a cysteine as the nucleophile, but a serine to coordinate the histidine base.[38][39] Despite the serine being a poor acid, it is still effective in orienting the histidine in the catalytic triad.[38] Some homologues alternatively have a threonine instead of serine at the acid location.[38]

Thr-Nter, Ser-Nter and Cys-Nter

Threonine proteases, such as the

penicillin acylase V[k] which are evolutionarily related to the proteasome proteases. Again, these use their N-terminal amide as a base.[26]

Ser-cisSer-Lys

This unusual triad occurs only in one superfamily of amidases. In this case, the lysine acts to polarise the middle serine.[40] 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 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.[40][41]

Sec-His-Glu

The rare, but naturally occurring amino acid selenocysteine (Sec), can also be found as the nucleophile in some catalytic triads.[29] Selenocysteine is similar to cysteine, but contains a 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.[29]

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.[42] Catalytic triads have also been inserted into otherwise non-catalytic proteins, or protein mimics.[citation needed]

Subtilisin (a serine protease) has had its oxygen nucleophile replaced with each of sulfur,[43][44] selenium,[45] or tellurium.[46] Cysteine and selenocysteine were inserted by mutagenesis, whereas the non-natural amino acid, tellurocysteine, was inserted using auxotrophic cells fed with synthetic tellurocysteine. These elements are all in the 16th periodic table column (chalcogens), so have similar properties.[47][48] In each case, changing the nucleophile reduced the enzyme's protease activity, but increased a different activity. A sulfur nucleophile improved the enzymes transferase activity (sometimes called subtiligase). Selenium and tellurium nucleophiles converted the enzyme into an oxidoreductase.[45][46] 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.[49]

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,[50] as well as a range of other proteins.[51] Similarly, catalytic triad mimics have been created in small organic molecules like diaryl diselenide,[52][53] and displayed on larger polymers like Merrifield resins,[54] and self-assembling short peptide nanostructures.[55]

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.[56] 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 adapted to serve different functions.[57][58] Some proteins, called pseudoenzymes, have non-catalytic functions (e.g. regulation by inhibitory binding) and have accumulated mutations that inactivate their catalytic triad.[59]

Reaction changes

Catalytic triads perform

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.[60] 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.[35][61] 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.[35]

Additionally, an alternative transferase mechanism has been evolved by

internal tunnel in the enzyme to the second active site, where it is transferred to a second substrate.[62][63]

Nucleophile changes

PA clan proteases to use different nucleophiles in their catalytic triad. Shown are the serine triad of chymotrypsin[e] and the cysteine triad of TEV protease.[a] (PDB: 1LVM, 1GG6
​)

Divergent evolution of active site residues is slow, due to strong chemical constraints. Nevertheless, some protease superfamilies have evolved from one nucleophile to another. This can be inferred when a superfamily (with the same fold) contains families that use different nucleophiles.[49] Such nucleophile switches have occurred several times during evolutionary history, however the mechanisms by which this happen are still unclear.[17][49]

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).

Superfamilies containing a mixture of families that use different nucleophiles [64]
Superfamily Families Examples
PA clan
 C3, C4, C24, C30, C37, C62, C74, C99 TEV protease (Tobacco etch virus)
S1, S3, S6, S7, S29, S30, S31, S32, S39, S46, S55, S64, S65, S75
Bos taurus
)
PB clan C44, C45, C59, C69, C89, C95
Homo sapiens
)
S45, S63
Penicillin G acylase precursor (Escherichia coli
)
T1, T2, T3, T6 Archaean proteasome, beta component (Thermoplasma acidophilum)
PC clan C26, C56
Rattus norvegicus
)
S51 Dipeptidase E (Escherichia coli)
PD clan C46
Hedgehog protein (Drosophila melanogaster
)
N9, N10, N11
Intein-containing V-type proton ATPase catalytic subunit A (Saccharomyces cerevisiae
)
PE clan P1 DmpA aminopeptidase (Brucella anthropi)
T5 Ornithine acetyltransferase precursor (Saccharomyces cerevisiae)

Pseudoenzymes

A further subclass of catalytic triad variants are

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).[70]

Superfamilies containing pseudoenzymes with inactive triads [65]
Superfamily Families containing pseudoenzymes Examples
CA clan C1, C2, C19
Calpamodulin
CD clan C14 CFLAR
SC clan S9, S33 Neuroligin
SK clan S14 ClpR
SR clan S60 Serotransferrin domain 2
ST clan S54 RHBDF1
PA clan
 S1 Azurocidin 1
PB clan T1 PSMB3

Convergent evolution

prolyl oligopeptidase,[n] TEV protease,[a] and papain.[c] (PDB: 1ST2, 1LVM, 3EQ8, 1PE6​)
Evolutionary convergence of threonine proteases towards the same N-terminal active site organisation. Shown are the catalytic threonine of the proteasome[h] and ornithine acetyltransferase.[i] (PDB: 1VRA, 1PMA
​)

The

superfamilies. Each of these superfamilies is the result of convergent evolution for the same triad arrangement within a different 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.[1][2]

Cysteine and serine hydrolases

The same triad geometries been converged upon by serine proteases such as the

C3 protease and papain[c] superfamilies. These triads have converged to almost the same arrangement due to the mechanistic similarities in cysteine and serine proteolysis mechanisms.[2]

Families of cysteine proteases

Superfamily Families Examples
CA C1, C2, C6, C10, C12, C16, C19, C28, C31, C32, C33, C39, C47, C51, C54, C58, C64, C65, C66, C67, C70, C71, C76, C78, C83, C85, C86, C87, C93, C96, C98, C101
Homo sapiens
)
CD C11, C13, C14, C25, C50, C80, C84
Rattus norvegicus) and separase (Saccharomyces cerevisiae
)
CE C5, C48, C55, C57, C63, C79
adenovirus
type 2)
CF C15 Pyroglutamyl-peptidase I (Bacillus amyloliquefaciens)
CL C60, C82 Sortase A (Staphylococcus aureus)
CM C18 Hepatitis C virus peptidase 2 (hepatitis C virus)
CN C9
Sindbis virus-type nsP2 peptidase (sindbis virus
)
CO C40 Dipeptidyl-peptidase VI (Lysinibacillus sphaericus)
CP C97
Mus musculus
)
PA
 C3, C4, C24, C30, C37, C62, C74, C99 TEV protease (Tobacco etch virus)
PB C44, C45, C59, C69, C89, C95
Homo sapiens
)
PC C26, C56
Rattus norvegicus
)
PD C46
Hedgehog protein (Drosophila melanogaster
)
PE P1 DmpA aminopeptidase (Brucella anthropi)
unassigned C7, C8, C21, C23, C27, C36, C42, C53, C75

Families of serine proteases

Superfamily Families Examples
SB S8, S53 Subtilisin (Bacillus licheniformis)
SC S9, S10, S15, S28, S33, S37
Sus scrofa
)
SE S11, S12, S13 D-Ala-D-Ala peptidase C (Escherichia coli)
SF S24, S26 Signal peptidase I (Escherichia coli)
SH S21, S73, S77, S78, S80 Cytomegalovirus
herpesvirus
5)
SJ S16, S50, S69
Lon-A peptidase (Escherichia coli
)
SK S14, S41, S49 Clp protease (Escherichia coli)
SO S74 Phage GA-1 neck appendage CIMCD self-cleaving protein (
Bacillus phage GA-1
)
SP S59
Homo sapiens
)
SR S60
Homo sapiens
)
SS S66 Murein tetrapeptidase LD-carboxypeptidase (Pseudomonas aeruginosa)
ST S54 Rhomboid-1 (Drosophila melanogaster)
PA
 S1, S3, S6, S7, S29, S30, S31, S32, S39, S46, S55, S64, S65, S75
Bos taurus
)
PB S45, S63
Penicillin G acylase precursor (Escherichia coli
)
PC S51 Dipeptidase E (Escherichia coli)
PE P1 DmpA aminopeptidase (Brucella anthropi)
unassigned S48, S62, S68, S71, S72, S79, S81

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.[2] When the nucleophile of a serine protease was mutated to threonine, the methyl occupied a mixture of positions, most of which prevented substrate binding.[71] Consequently, the catalytic residue of a threonine protease is located at its N-terminus.[2]

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)[32] and Superfamily PE (acetyltransferases using the DOM fold)[33] This commonality of active site structure in completely different protein folds indicates that the active site evolved convergently in those superfamilies.[2][26]

Families of threonine proteases

Superfamily Families Examples
PB clan T1, T2, T3, T6 Archaean proteasome, beta component (Thermoplasma acidophilum)
PE clan T5 Ornithine acetyltransferase (Saccharomyces cerevisiae)

See also

References

Notes

  1. ^ a b c d TEV protease MEROPS: clan PA, family C4
  2. ^ a b Cytomegalovirus protease MEROPS: clan SH, family S21
  3. ^ a b c d Papain MEROPS: clan CA, family C1
  4. ^ Hepatitis A virus protease MEROPS: clan PA, family C3
  5. ^ a b c Chymotrypsin MEROPS: clan PA, family S1
  6. ^ Sedolisin protease MEROPS: clan SB, family 53
  7. ^ Vasohibin protease MEROPS: clan CA
  8. ^ a b Proteasome MEROPS: clan PB, family T1
  9. ^ a b Ornithine acyltransferases MEROPS: clan PE, family T5
  10. ^ Penicillin acylase G MEROPS: clan PB, family S45
  11. ^ Penicillin acylase V MEROPS: clan PB, family C59
  12. ^ amidophosphoribosyltransferase MEROPS: clan PB, family C44
  13. ^ Subtilisin MEROPS: clan SB, family S8
  14. ^ Prolyl oligopeptidase MEROPS: clan SC, family S9

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