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68 Chapter 4
4.5 The existence of intermediates in enzymatic reactions
Enzymatic reactions are often multi-step reactions involving a number of
transient enzyme-bound intermediates (Figure 4.18). If the enzymatic reaction
is very rapid and none of the intermediates are released from the active site, how
can we prove the existence of these transient intermediates
Direct observation
Since the turnover numbers for most enzymes are > 1s 1 then it is impractical
to observe the formation of intermediates directly. However, in some cases the
turnover number can be reduced by changing the temperature or pH, or by
using an unnatural substrate, to such an extent that intermediates can be
detected directly by NMR spectroscopy.
If no intermediate is detectable by these methods then a faster analytic
method can be used in the form of stopped Xow methods. Just as stopped
Xow methods can be used to study rapid enzyme kinetics, in the same way rapid
quench methods can be used to isolate intermediates. This method involves
mixing enzyme with substrate in a rapid mixing device similar to that shown in
Figure 4.8, then, after a Wxed time interval of say 100 ms, mixing with a quench
reagent such as an organic solvent or a diVerent pH solution.
This type of approach was recently used to identify a tetrahedral intermediate
in the reaction of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, as
shown in Figure 4.19 (also see Section 8.5). In this case the intermediate was
isolated by quenching 50 mg quantities of enzyme and substrate with neat
triethylamine, which was found to stabilise the intermediate.
Trapping
Intermediates in enzymatic reactions that possess enhanced chemical reactivity
can sometimes be trapped using a selective chemical reagent. One example
free
energy
E + S
ES
E.Int1 E.Int2
E.P
Figure 4.18 Energy proWle of a multi-step enzymatic reaction.
E + P
reaction co-ordinate