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.

[1][2] Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, lipases and β-lactamases).

[4][5] In the 1950s, a serine residue was identified as the catalytic nucleophile of trypsin and chymotrypsin (first purified in the 1930s)[6] by diisopropyl fluorophosphate modification.

[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.

[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.

[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.

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.

The resulting build-up of negative charge is typically stabilized by an oxyanion hole within the active site.

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[25] has been controverted.

The lone pair of 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.

Compared to oxygen, sulfur's extra d orbital makes it larger (by 0.4 Å)[29] 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).

[30] Serine is therefore more dependent than cysteine on optimal orientation of the acid-base triad members to reduce its pKa[30] 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.

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.

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.

[1] The second histidine is not as effective an acid as the more common aspartate or glutamate, leading to a lower catalytic efficiency.

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).

[35][36] 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.

The middle serine is held in an unusual cis orientation to facilitate precise contacts with the other two triad residues.

[45][46] The rare, but naturally occurring amino acid selenocysteine (Sec), can also be found as the nucleophile in some catalytic triads.

[57] Similarly, catalytic triad mimics have been created in small organic molecules like diaryl diselenide,[58][59] and displayed on larger polymers like Merrifield resins,[60] and self-assembling short peptide nanostructures.

[63][64] Some proteins, called pseudoenzymes, have non-catalytic functions (e.g. regulation by inhibitory binding) and have accumulated mutations that inactivate their catalytic triad.

Several families of transferase enzymes have evolved from hydrolases by adaptation to exclude water and favour attack of a second substrate.

[k] In the first active site, a cysteine triad hydrolyses a glutamine substrate to release free ammonia.

[68][69] Divergent evolution of active site residues is slow, due to strong chemical constraints.

These examples reflect the intrinsic chemical and physical constraints on enzymes, leading evolution to repeatedly and independently converge on equivalent solutions.

[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.

The enzyme TEV protease [ a ] contains an example of a catalytic triad of residues (red) in its active site . The triad consists of an 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 charge-relay system as commonly found in proteases. The acid residue (commonly glutamate or aspartate ) aligns and polarises the base (usually histidine ) which activates the nucleophile (often serine or cysteine, occasionally threonine ). The triad reduces the p K a 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 .
The range of amino acid residues found at the active sites of hydrolytic enzymes. On the left are the nucleophile, base and acid triad members. On the right are substrates with the cleavable bond indicated by a pair of scissors. Two bonds in beta-lactams can be cleaved (1 by penicillin acylase and 2 by beta-lactamase ).
Divergent evolution of PA clan proteases to use different nucleophiles in their catalytic triad. Shown are the serine triad of chymotrypsin [ c ] and the cysteine triad of TEV protease. [ a ] ( PDB : 1LVM , 1GG6 ​)