A catalytic triad refers to the three amino acid residues that function together at the centre of the active site of some hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, lipases and β-lactamases). An Acid-Base-Nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to regenerate free enzyme. The nucleophile is most commonly serine or cysteine but occasionally threonine.
Because enzymes fold into complex three-dimensional structures, the residues of a catalytic triad can be far from each other along the amino-acid sequence (primary structure), however, they are brought close together in the final fold.
As well as divergent evolution of function (and even the triad's nucleophile), catalytic triads show some of the best examples of convergent evolution. Chemical constraints on catalysis have led to the same catalytic solution independently evolving in at least 23 separate superfamilies. Their mechanism of action is consequently one of the best studied in biochemistry.
- 1 History
- 2 The identity of triad members
- 3 Examples of triads
- 4 Comparison of serine and cysteine hydrolase mechanisms
- 5 Divergent evolution
- 6 Convergent evolution
- 7 See also
- 8 References
The structures of trypsin and chymotrypsin were first solved in the 1930s. The serine triad member of trypsin and chymotrypsin was identified as the nucleophile (by diisopropyl fluorophosphate modification) in the 1950s. Other protease sequences were aligned in the 1960s to reveal a family of related proteases, now called the S1 family. Simultaneously, the structures of the evolutionarily unrelated papain and subtilisin 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 in the 1970s and 80s, homologous (such as TEV protease) and analogous (such as papain) triads were found. The MEROPS classification system in the 1990s and 2010s started to class proteases into structurally related enzyme superfamilies and so acts as a database of the convergent evolution of triads in over 20 superfamilies. Understanding of the chemical constraints on evolution that have led to the convergence of so many enzyme families on the same triad geometries has begun to be understood in the 2010s. The massive body of work on the charge-relay, covalent catalysis of catalytic triads has led to the mechanism as being the best characterised in all of biochemistry.
The identity of triad members
The side-chain of the nucleophilic residue performs covalent catalysis on the substrate. The lone pair of electrons present on the oxygen or sulphur attack the electropositive carbonyl carbon. The 20 naturally occurring biological amino acids do not contain sufficiently nucleophilic functional groups for many difficult catalytic reactions. The most commonly used nucleophiles are the alcohol (OH) of serine and the thiol/thiolate ion (SH/S−) of cysteine. Embedding the nucleophile in a triad makes it more catalytically active. A few proteases use the secondary alcohol of threonine, however, due to the extra methyl group, such proteases use the N-terminal amide as the base, rather than a separate amino acid.
Since no natural amino acids are strongly nucleophilic, the base in a catalytic triad polarises and deprotonates the nucleophile to increase its reactivity. Additionally, it protonates the first product to aid leaving group departure. It is most commonly histidine since its pKa allows for effective base catalysis as well as both hydrogen bonding to the acid residue and deprotonating the nucleophile residue. β-lactamases such as 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. In order to avoid steric clashes, threonine proteases use their N-terminal amide as the base, to increase the reactivity of the catalytic threonine residue.
The acidic residue aligns and polarises the basic residue. It is commonly aspartate or glutamate. Some enzymes act only as a dyad as the acid member of the triad can be less necessary for cysteine proteases. For example papain uses asparagine as its third triad member which orients the histidine base but cannot act as an acid. Similarly, hepatitis A virus protease contains an ordered water in the position where an acid residue should be. Lastly, cytomegalovirus proteases 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.
Examples of triads
Chymotrypsin (Superfamily PA, Family S1) is considered as one of the classic triad-containing enzymes. It uses a Serine-Histidine-Aspartate motif for proteolysis. Chymotrypsin binds its substrate, an exposed loop containing a large hydrophobic residue. The aspartate is hydrogen bonded (possibly low-barrier hydrogen bond) with histidine, increasing the pKa of its imidazole nitrogen from 7 to about 12. This allows the histidine to act as a powerful general base, and deprotonate serine. The serine serves as a nucleophile, attacking the carbonyl carbon and forcing the carbonyl oxygen to accept an electron, leading to a tetrahedral intermediate. This intermediate is stabilized by an oxanion hole, involving the backbone amide of serine. Collapse of this intermediate back to a carbonyl causes histidine to donate its proton to the nitrogen attached to the alpha carbon. The nitrogen and the attached C-terminal peptide fragment leave by diffusion. A water molecule then donates a proton to histidine and the remaining OH− attacks the carbonyl carbon, forming another tetrahedral intermediate. The OH is a poorer leaving group than the C-terminal fragment, so, when the tetrahedral intermediate collapses again, the enzyme's serine leaves, regaining a proton from histidine. The N-terminus of the cleaved peptide now leaves by diffusion. The same triad has also convergently evolved in α/β hydrolases such as some lipases and esterases, however the chirality is reversed. Additionally, brain acetyl hydrolase (which has the same fold as a small G-protein) has also been found to have this triad. The equivalent Serine-Histidine-Glutamate triad is used in acetylcholinesterase.
Several families of cysteine proteases use this triad set, for example TEV protease (Superfamily PA, Family C4) and papain (Superfamily CA, Family C1). The triad acts similarly to serine protease triads, with notable differences discussed in 'Comparison of serine and cysteine hydrolase mechanisms'. It is still unclear how important the Asp of the papain triad is to catalysis and several cysteine proteases are effectively dyads (e.g. hepatitis A virus protease).
The triad of cytomegalovirus protease (Superfamily SH, Family S21) uses histidine as both the acid and base triad members. Removing the acid histidine only results in 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.
An unusual triad is found in seldolisin proteases (Superfamily SB, Family S53). The low pKa 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 to specific environments like acidic hot springs (e.g. kumamolysin) or cell lysosome (e.g. tripeptidyle peptidase).
Threonine proteases, such as the proteasome protease subunit (Superfamily PB, Family T1) and ornithine acyltransferases (Superfamily PE, Family T5) use the secondary alcohol of threonine in an manner analogous to the use of the serine primary alcohol. 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 alcohol to increase its reactivity.
Ser-Nterm and Cys-Nterm
In a similar manner to threonine proteases, there exist equivalent 'serine only' and 'cysteine only' configurations such as penicillin acylase G (Superfamily PB, Family S45) and penicillin acylase V (Superfamily PB, Family S59) which are evolutionarily related to the proteasome proteases. Again, these use their N-terminal amide as a base.
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 he nucleophilic serine to activate it (one with the side chain alcohol 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 cisserine 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.
Comparison of serine and cysteine hydrolase mechanisms
Nucleophilic enzymes use an interconnected set of active site residues to achieve catalysis. The sophistication of the active site network causes residues involved in catalysis, and residues in contact with these, to be the most evolutionarily conserved within their families. In catalytic triads, the most common nucleophiles are serine (an alcohol) or cysteine (a thiol). Compared to oxygen, sulphur’s extra d orbital makes it larger (by 0.4 Å), softer, form longer bonds (dC-X and dX-H by 1.3-fold) and have lower pKa (by 5 units). Here I concentrate on chemical differences between cysteine and serine proteases on catalytic chemistry, however similar issues affect hydrolases and transferases in general.
The pKa of cysteine is low enough that some cysteine proteases (e.g. papain) have been shown to exist as an S− thiolate ion in the ground state enzyme (a) and many even lack the acidic triad member (b). Serine is also more dependent on other residues to reduce its pKa for concerted deprotonation with catalysis (c) by optimal orientation of the acid-base triad members (d). The low pKa of cysteine works to its disadvantage in the resolution of the first tetrahedral intermediate as unproductive reversal of the original nucleophilic attack is the more favourable breakdown product. The triad base is therefore preferentially oriented to protonate the leaving group amide (e) to ensure that it is ejected to leave the enzyme sulphur 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 (f) whereas cysteine can leave as S−.
Sterically, the sulphur of cysteine also has longer bonds and a bulkier Van der Waals radius to fit in the active site and a mutated nucleophile can be trapped in unproductive orientations. For example the crystal structure of thio-trypsin indicates that cysteine points away from the substrate, instead forming interactions with the oxyanion hole.
The evolutionary specialisation of enzymes around the needs of their nucleophile makes it unsurprising that nucleophiles cannot be interconverted in extant proteases (nor in most other enzymes) and the large activity reductions (>104) observed can be explained as a result of compromised reactivity or structural misalignment.
Despite the chemical differences described above, it is clear that some protease superfamilies have evolved to use different nucleophiles through divergent evolution. This can be inferred because of several superfamilies (with the same fold) contain families that use different nucleophiles, indicating that nucleophile switches have occurred several times during evolutionary history, however the evolutionary mechanisms by which this can happen are still unclear.
|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||Chymotrypsin (mammals, e.g. bos taurus)|
|PB clan||C44, C45, C59, C69, C89, C95||amidophosphoribosyltransferase precursor (Homo sapiens)|
|S45, S63||penicillin G acylase precursor (Escherichia coli)|
|T1, T2, T3, T6||archaean proteasome, beta component (Thermoplasma acidophilum)|
|PC clan||C26, C56||gamma-glutamyl hydrolase (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 (Ochrobactrum anthropi)|
|T5||Ornithine acetyltransferase precursor (Saccharomyces cerevisiae)|
The enzymology of proteases provides some of the clearest examples of convergent evolution. The same geometric arrangement of triad residues have independently evolved over 20 times (in separate enzyme superfamilies). 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 constraints on enzymes, leading evolution to independently converge on equivalent solutions repeatedly.
Cysteine and serine hydrolases
Serine and cysteine proteases use different amino acid functional groups (alcohol or thiol) as a nucleophile. In order to activate that nucleophile, they orient an acidic and basic residue in a catalytic triad. The chemical and physical constraints on enzyme catalysis have caused identical triad arrangements to have evolved independently over 20 times in different enzyme superfamilies.
The same triad geometries been converged upon by serine proteases such as chymotrypsin and subtilisin superfamilies. Similarly, the same has occurred with cysteine proteases such as viral C3 protease and papain superfamilies. Importantly, due to the mechanistic similarities in cysteine and serine proteases, all of these triads have converged to almost the same arrangement.
Threonine proteases use the amino acid threonine as their catalytic nucleophile. Unlike cysteine and serine, threonine is a secondary alcohol (i.e. has a methyl group). The methyl group of threonine greatly restricts the possible orientations of triad and substrate as the methyl clashes with either the enzyme backbone or histidine base. Consequently, most threonine proteases use an N-terminal threonine in order to avoid such steric clashes.
Several evolutionarily independent enzyme superfamilies with different protein folds use the N-terminal residue as a nucleophile. Firstly they occurs in Superfamily PB (proteosomes using the Ntn fold) and secondly in Superfamily PE (acetyltransferases using the DOM fold) This commonality of active site in completely different protein folds indicates that the active site evolved convergently in those superfamilies.
|Superfamily||Threonine protease families||Examples|
|PB clan||T1, T2, T3, T6||archaean proteasome, beta component (Thermoplasma acidophilum)|
|PE clan||T5||ornithine acetyltransferase (Saccharomyces cerevisiae)|
- Enzyme catalysis
- Functional groups
- Enzyme superfamily
- PA clan
- Convergent evolution
- Divergent evolution
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