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204 Chapter 8
involves no change in redox level. Recent experiments have indicated that the
Xavin cofactor is involved somehow in the elimination reaction, since incubation
of the 6R-6-Xuoro analogue (in which the proton removed is substituted
with Xuorine) with enzyme leads to the observation of a Xavin semiquinone
species. It, therefore, appears that one-electron transfers may be involved in the
mechanism employed by this enzyme.
8.5 CASE STUDY: 5-Enolpyruvyl-shikimate-3-phosphate
(EPSP) synthase
There are a number of enzymes that catalyse multi-step addition-elimination
reactions, of which the best characterised is the enzyme EPSP synthase. This
enzyme catalyses the sixth step on the shikimate pathway, namely the transfer
of an enolpyruvyl moeity from phosphoenol pyruvate (PEP) to shikimate-
3-phosphate (see Figure 8.1).
Early experiments on the mechanism of this enzyme examined 3 H exchange
processes. Incubation of [3- 3 H]-PEP with enzyme and shikimate-3-phosphate
led to the release of 3 H label into solvent, consistent with the existence of an
intermediate containing a rotatable methyl group, as shown in Figure 8.18.
Subsequent overexpression of this enzyme allowed a detailed examination
of the reaction using stopped Xow kinetics and rapid quench methods. Analysis
of a single turnover of the enzymatic reaction by stopped Xow methods revealed
the existence of an intermediate formed from shikimate-3-phosphate and PEP
after 5 ms and eventually consumed after 50 ms. Incubation of large amounts of
enzyme with shikimate-3-phosphate and PEP followed by rapid quench of the
enzymatic reaction into 100% triethylamine led to the isolation of small
amounts of the desired intermediate. This substance was a good substrate for
both the forward and reverse reactions of EPSP synthase, satisfying the criteria
for establishment of an intermediate in an enzymatic reaction.
Large-scale rapid quench studies using 500 mg of pure enzyme gave 300 mg
of intermediate, which was characterised by 1 H, 13 Cand 31 P nuclear magnetic
CO 2
−
3 H
2− O 3 PO
CO 2
−
OH
OH
+
2− O 3 PO
3 H
CO 2
−
2− O 3 PO
CO 2
−
OH
H H 3 H
−
O CO 2
2−
OPO 3
loss of H +
FAST
2− O 3 PO
OH
CO 2
−
O
+H + + P i
−
CO 2
loss of 3 H +
SLOW
2− O 3 PO
O
CO 2
−
+ 3 H + + P i
OH
release of 3 H
into solvent
Figure 8.18 3 H exchange in the EPSP synthase reaction.