Catalytic triad

An enzyme (TEV protease 1lvm) bound to a substrate (black) contains a catalytic triad of residues (red). The triad consists of an acidic residue (Acid), histidine (His) and the nucleophile (Nuc). In the case on TEV protease, the acid is aspartate and the nucelophile is cysteine.

A catalytic triad usually refers to the three amino acid residues that function together at the centre of the active site of certain hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, lipases and β-lactamases). A common method for generating a nucleophilic residue for covalent catalysis is by using an Acid-Base-Nucleophile triad,. 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. They nucleophile is most commonly serine or cysteine but occasionally threonine.

Because enzymes fold into complex three-dimensional shapes, the residues of a catalytic triad can be far from each other in the along the primary structure amino-acid sequence; however, they are brought close together when the chain folds into its 3-dimensional tertiary structure.

The identity of triad members

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 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 substrate. 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.

Base

The base in a catalytic triad 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.

Acid

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, hepatitus 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 is the more common aspartate or glutamate, leading to a lower catalytic efficiency.

Examples of triads

Ser-His-Asp

Chymotrypsin (Superfamily PA, Family S1) is considered as one of the classic triad-containing enzymes. It uses a Serine-Histidine-Aspartate motif for proteolysis.

1. Upon binding of the target protein, the carboxylic group (-COOH) on D102 forms a low-barrier hydrogen bond with H57, increasing the pKa of its imidazole nitrogen from 7 to about 12. This allows H57 to act as a powerful general base, and deprotonate S195.
2. The deprotonated S195 serves as a nucleophile, attacking the carbonyl carbon on the C-terminal side of the residue and forcing the carbonyl oxygen to accept an electron, and transforming the sp2 carbon into a tetrahedral intermediate. This intermediate is stabilized by an oxanion hole, which also involves S195.
3. Collapse of this intermediate back to a carbonyl causes H57 to donate its proton to the nitrogen attached to the alpha carbon. The nitrogen and the attached peptide fragment (c-terminal to the F W or Y residue) leave by diffusion.
4. A water molecule then donates a proton to H57 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, S195 leaves and regains a proton from H57.
5. The cleaved peptide, now with a carboxyl end, 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.

Cys-His-Asp

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 catalytic triad#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).

Ser-His-His

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.

Ser-Glu-Asp

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).

Thr-Nterm

Threonine proteases, such as archean proteasome (Superfamily PB, Family T1) use the alcohol of threonine in an analagous manner to the use of the serine alcohol. However, due to the steric interference of the extra methyyl goupr of threonine, the base member of the triad has to be the N-terminal amide functional group.

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 archean proteasome proteases. Again, these use their N-terminal amide as a base.

Ser-cisSer-Lys

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 wih 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

Differences in cysteine and serine proteolysis mechanisms.The protease (black) performs a nucleophilic attack on peptide substrate (red) to form a tetrahedral intermediate. This breaks down by ejection of the first product, the substrate C-terminus, to form the acyl-enzyme intermediate. Water replaces the first product and hydrolysis occurs via a second tetrahedral intermediate to regenerate free enzyme. Indicated differences are (a) the deprotonated cysteine, (b) aspartate (grey) not present in all cysteine proteases, (c) concerted deprotonation of serine, (d) aspartate hydrogen bonding, (e) amide protonation of the first leaving group, (f) alcohol protonation of the serine leaving group.

This section references research done on proteases, however the same mechanisms and arguments apply to serine and cysteine hydrolases in general.

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 (d_C-X and d_X-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.

Divergent evolution

Despite the chemical differences mentioned above, it is clear that some protease superfamilies have evolved to use different nucleophiles though 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.

Within the protease superfamilies containing a mixture of nucleophiles (e.g. the PA clan), families are designated by their catalytic nucleophile (C=cysteine proteases, S=serine proteases).

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	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)

Convergent evolution

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.

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	papain (Carica papaya) and calpain (Homo sapiens)
CD	C11, C13, C14, C25, C50, C80, C84	caspase-1 (Rattus norvegicus) and separase (Saccharomyces cerevisiae)
CE	C5, C48, C55, C57, C63, C79	adenain (human 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	DeSI-1 peptidase (Mus musculus)
PA	C3, C4, C24, C30, C37, C62, C74, C99	TEV protease (Tobacco etch virus)
PB	C44, C45, C59, C69, C89, C95	amidophosphoribosyltransferase precursor (Homo sapiens)
PC	C26, C56	gamma-glutamyl hydrolase (Rattus norvegicus)
PD	C46	hedgehog protein (Drosophila melanogaster)
PE	P1	DmpA aminopeptidase (Ochrobactrum anthropi)
unassigned	C7, C8, C21, C23, C27, C36, C42, C53, C75

Threonine proteases

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 methyly 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 occus in Superfamily PB (using the Ntn fold) and secondly in Superfamily PE (usind the DOM fold) This commonality of active site in completely different protein folds indicates that the active site evolved convergently in those superfamilies.

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

See also

• Functional groups
• Enzyme superfamily
• Enzyme catalysis
• PA clan
• Convergent evolution
• Divergent evolution

References

• Lehninger, Principles of Biochemistry, 4th ed.
• Wilson, Eisner, Briggs, Dickerson, Metzenberg, O'Brien, Susman, Boggs, Life on Earth (c 1973, Sinauer Associates, Inc., Publisher, Stamford, Connecticut. ISBN 0-87893-934-2)