A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes.[1][2] Catalytic triads are most commonly found in 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 covalentintermediate which is then 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 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.[2] Their mechanism of action is consequently one of the best studied in biochemistry.[4][5]
The enzymes trypsin and chymotrypsin were first purified in the 1930s.[6] A serine in each of trypsin and chymotrypsin was identified as the catalytic nucleophile (by diisopropyl fluorophosphate modification) in the 1950s.[7] The structure of chymotrypsin was solved by X-ray crystallography in the 1960s, showing the orientation of the catalytic triad in the active site.[8] Other proteases were sequenced and aligned to reveal a family of related proteases,[9][10][11] 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.[12]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.[13][14][15] The MEROPS 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.[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 low-barrier hydrogen bonding contributed to catalysis,[18][19][20] or whether ordinary 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]
The side-chain of the nucleophilic residue performs covalent catalysis on the substrate. The lone pairof electrons present on the oxygen or sulfur attacks the electropositive carbonyl carbon.[3] 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 (OH) of serine and the thiol/thiolate ion (SH/S−) of cysteine.[2] Alternatively, threonine proteases use the secondary hydroxyl of threonine, however due to steric hindrance of the side chain's extra methyl group such proteases use their 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 oxygen, sulfur's extra d orbital makes it larger (by 0.4 Ã…)[27] and softer, allows it to form longer bonds (dC-X and dX-H by 1.3-fold), and gives it a lower pKa (by 5 units).[28] Serine is therefore more dependent than cysteine on optimal orientation of the acid-base triad members to reduce its pKa[28] in order to achieve concerted deprotonation with catalysis.[2] 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.[2] The triad base is therefore preferentially oriented to protonate 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[2] and if 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]
The same triad has also convergently evolved in α/β hydrolasessuch as some lipases and esterases, however orientation of the triad members is reversed.[34][35] Additionally, brain acetyl hydrolase(which has the same fold as a small 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[edit]
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[edit]
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[edit]
An unusual triad is found in seldolisin proteases.[f] 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. tripeptidyl peptidase).[26]
Thr-Nter, Ser-Nter and Cys-Nter[edit]
Threonine proteases, such as the proteasome protease subunit[h] and ornithine acyltransferases[i] use the secondary hydroxyl of threonine in a manner analogous to the use of the serine primary hydroxyl.[32][33] 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.[1][26]Similarly, there exist equivalent 'serine only' and 'cysteine only' configurations such as penicillin acylase G[j] and 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[edit]
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[edit]
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[edit]
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 peptidenanostructures.[55]
| 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 | Chymotrypsin (mammals, e.g. Bos taurus) | |
| PB clan | C44, C45, C59, C69, C89, C95 | Amidophosphoribosyltransferaseprecursor (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 acetyltransferaseprecursor (Saccharomyces cerevisiae) |
https://en.wikipedia.org/wiki/Catalytic_triad
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