Some of the information about the acyl enzyme comes from investigations in which the substrate supplied was an ester, rather than a polypeptide.

The reason for this is that the esters are more reactive in the initial nucleophilic attack, so the deacylation typically becomes the rate determining step.

These esters have the advantage that the leaving group is a chromophore (p-nitrophenoxide) making it easy to follow the kinetics of the hydrolysis by spectrophotometry.

• Following the release of phenoxide as a function of time, and extrapolating back to zero time, investigators found that the extrapolation did not lead to zero concentration

• Instead, an initial "burst" occurred: phenoxide was released instantaneously on their time scale in concentration equal to the concentration of enzyme.

The implication:

• The ester rapidly acylates the enzyme mole for mole

• Deacylation is the slow step

This is inferential evidence. It implies an intermediate, but does not identify the intermediate

• It could possibly arise instead by the substrate inducing a conformational change in the enzyme upon binding.

• Stopped-flow measurements demonstrated that both acylation and deacylation were at least equal to the overall k_cat
  • This supports the view that the "burst" comes from accumulation of an acyl intermediate.

If several different substrates for the enzyme generate the same acyl enzyme intermediate, and its breakdown is the rate determining step, then all should hydrolyze with the same k_cat.

These data imply that a common intermediate is formed, and by inference, since the common part of the structures is the acyl group, that it is the acyl enzyme.

As usual, some of the best evidence is crystallographic; the acyl enzymes are stable enough to be crystallized, especially when the acyl group is not a peptide.

Guanidinobenzoyl Trypsin (1gbt)

Here are a couple more examples:

Cinnamoyl Subtilisin (1be8)	Elastase with Casomorphin (YPFVEPI) (1hax)

At this point the protein has been cleaved, but half of it is now covalently bonded to the enzyme.

Now we must deacylate. To do so, we need a water molecule; one is found in an appropriate location in the subtilisin crystal structure above: