The attack of water produces another tetrahedral intermediate, just as in the solution hydrolysis of an ester, which is now the analogous in vitro reaction.
The transfer of the proton from the His back to the Ser is probably a separate step, following so swiftly that it cannot be distinguished.
This tetrahedral intermediate has been trapped for the reaction of elastase with casomorphin by soaking the crystal of the acyl enzyme in a buffer at pH 9 and then flash freezing it in liquid nitrogen [Schofield, et al, Nat. Struct. Biol., 2001, 8, 689]:
| Tetrahedral Intermediate, Elastase and Casomorphin (1haz) |
|---|
 |
All that remains is to release the carboxylic acid. The enzyme is restored to its original state, ready for another catalytic event.
When deacylation is the rate-determining step, kcat should depend upon the structure of the R in the acyl group.
| R in R(C=O)-OEnz | kcat | kin vitro |
|---|---|---|
| CH3 | 0.01 | 0.19 |
| PhCH2CH2 | 0.178 | 0.15 |
| CH3CONH-CH2 | 0.12 | 2.48 |
| S-PhCH2CH(NHCOCH3) | 111 | 1.94 |
| Enz = chymotrypsin | ||
Chymotrypsin, remember, has the binding pocket that fits the phenyl group tightly; note
This latter binding has been suggested to involve a hydrogen bond between the NH of the amide bond in the substrate and the backbone C=O of Ser214:
An interesting point, stimulated by the observation above about Ser214. An attempt was made to use site-directed mutation to give trypsin the activity of chymotrypsin. Here are the mutations and the results:
Here's a picture of chymotrypsin with the residues of that last item highlighted in violet (the catalytic center is green):
Implication: the active site pocket is not the only determinant of selectivity.
The three extra regions delineated above stretch out across the surface of the enzyme, and perhaps help to position the substrate so that the aromatic side chain can "find" the pocket.