User:Evolution and evolvability/sandbox

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 found at the centre of the active site of certain enzymes including hydrolases and transferases. A common method for generating a nucleophilic residue for catalysis is by using an Acid-Base-Nucleophile triad21,22. 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

In general terms, catalytic triad can refer to any set of three residues that function together and are directly involved in catalysis. 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.

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 families58. 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 Å229), softer, form longer bonds (d_C-X and d_X-H by 1.3-fold) and have lower pKa (by 5 units230). 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 enzyme231 (a) and many even lack the acidic triad member (b). Serine is also more dependent on other residues to reduce its pKa230 for concerted deprotonation with catalysis (c) by optimal orientation of the acid-base triad members (d)21. 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 product21. 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 site232 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 hole229.

The evolutionary specialisation of enzymes around the needs of their nucleophile makes it unsurprising that nucleophiles cannot be interconverted in extant proteases211,231,233–237 (nor in most other enzymes238–243) and the large activity reductions (>10⁴) observed can be explained as a result of compromised reactivity or structural misalignment.

Example

An example of a catalytic triad is present in chymotrypsin, wherein the triad (on the enzyme) consists of S195 (that is, the serine found at residue 195 in the protein sequence), D102 and H57. In essence, S195 binds to the substrate polypeptide to the side of a phenylalanine, tryptophan, or tyrosine residue (the residue is on the C-terminus side), holding it in place. D102 and H57 then hydrolyze the bond. This takes place in several steps.

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.

Amino acid sequence, histidine 57

For three example proteases, chymotrypsin A (cow), trypsin (cow), and elastase (pig), the amino acid residues are listed in the following table; the (vertical column)-histidine 57, is coded as capital H:
(see: Sequence of proteases - chymotrypsin A - trypsin - elastase)

A few sections are shown as very similar, and likewise sections that are quite dissimilar-(the sections away from the "active 3-dimensional site"). From the residues 46-99A, the most common area is from residue 46 to 58, one residue past residue Histidine 57; it is even more similar, from the residues 51-58.

x	46-50	51 - 55	56,57-60	65	65A 66-70
C	LINEN	WVVTA	AHCGV	TTSDV	'VVAGEFD
T	LINSQ	WVVSA	AHCYK	SGIQV	RL'`GQDN
E	LIRQN	WVMTA	AHCVD	RELTF	RVVVGEHN

x	73-75-80	85	90	95	96-99A
C	QGSSSEKI	QKLKI	AKVFK	NSKYN	SLTI'
T	INVVEGNQ	QFISA	SKSIV	HPSYN	SNTL'
E	LNQNNGTE	QYVGV	QKIVV	HPYWN	TDDVA

Four atom characterize different ASP-HIS-SER enzyme families

Distinct concave regions correspond to different enzyme families, containing the catalytic triad ASP-HIS-SER. The relative positions of only the four, red-circled atoms well characterize whole enzyme families

By scanning the whole Protein Data Bank, containing the 3D structures of proteins, for the presence of the catalytic triad, one can find several hundred protein structures with the triad,. It is interesting that the relative positions of just four atoms (circled by red on the figure to the right) well characterize different triad-containing enzyme families

See also

• functional groups

References

• Lehninger, Principles of Biochemistry, 4th ed. (pp 216–219)
• Wilson, Eisner, Briggs, Dickerson, Metzenberg, O'Brien, Susman, & Boggs. Life on Earth, Edward O. Wilson, Thomas Eisner, Winslow R. Briggs, Richard E. Dickerson, Robert L. Metzenberg, Richard D. O'Brien, Millard Susman, William E. Boggs, c 1973, Sinauer Associates, Inc., Publisher, Stamford, Connecticut. 1033 pp, 19 p Index & Back Page (hardcover, ISBN 0-87893-934-2)