Catalytic triad
A catalytic triad is a set of three coordinated amino acid residues that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. 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 release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine, 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.
As well as divergent evolution of function, 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.
History
In the 1950s, a serine residue was identified as the catalytic nucleophile of trypsin and chymotrypsin by diisopropyl fluorophosphate modification. The structure of chymotrypsin was solved by X-ray crystallography 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 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 and analogous triads were found. 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. 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.Researchers have since conducted increasingly detailed investigations of the triad's 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 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 a substrate or to transfer one portion of a substrate over to a second substrate. Triads are an inter-dependent set of residues in the active site of an enzyme and act in concert with other residues to achieve 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.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.Catalysis is performed in two stages. First, the activated nucleophile attacks the carbonyl carbon and forces the carbonyl oxygen to accept an electron pair, leading to a tetrahedral intermediate. The resulting build-up of negative charge is typically stabilized by an 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. Although general-acid catalysis for breakdown of the First and Second tetrahedral intermediate may occur by the path shown in the diagram, evidence supporting such a mechanism with chymotrypsin has been controverted.
The second stage of catalysis is the resolution of the acyl-enzyme intermediate by the attack of a second substrate. If the substrate is water then hydrolysis results; if it is an organic molecule then that molecule is transferred onto the first substrate. Attack by the 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. The lone pair of electrons present on the oxygen or sulfur attacks the electropositive 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 of serine and the thiol/thiolate ion of cysteine. 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.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 and softer, allows it to form longer bonds, and gives it a lower pKa. Serine is therefore more dependent than cysteine on optimal orientation of the acid-base triad members to reduce its pKa in order to achieve concerted deprotonation with catalysis. 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 to ensure that it is ejected to leave the enzyme sulfur covalently bound to the substrate N-terminus. Finally, resolution of the acyl-enzyme 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 and if mutated to serine can be trapped in unproductive orientations in the active site.
Very rarely, the selenium atom of the uncommon amino acid 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 deprotonates the nucleophile to increase its reactivity. Additionally, it protonates the first product to aid leaving group departure.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. β-lactamases such as TEM-1 use a lysine residue as the base. Because lysine's pKa is so high, 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, leading to mutual alignment via restriction of the basic residue's side-chain rotation. The positive charge on the basic residue is simultaneously stabilised, leading to its polarisation. Two amino acids have acidic side chains at physiological pH and so are the most common members of the acidic triad residue. Cytomegalovirus 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.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, 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 pKa of its imidazole nitrogen from 7 to around 12. Histidine is thus able to act as a powerful general base, activating the serine nucleophile. 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 lipases and esterases, however orientation of the triad members is reversed. Additionally, brain acetyl hydrolase has also been found to have this triad.