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Methods for Studying Enzymatic Reactions 69
CO 2
−
2− O 3 PO
OH
+
2− O 3 PO
CO 2
−
EPSP
synthase
OH
CO 2
−
CO 2
−
CH 3
2− O 3 PO
OH
O
2−
OPO 3
CO
−
2
2− O 3 PO
OH
O
CO 2
−
isolated by rapid quench in 100% NEt 3 !
Figure 4.19 IdentiWcation of 5-endpyruvyl-shikimate-3-phosphate (EPSP) synthase reaction
intermediate. NEt 3 , triethylamine.
substrate
X-Enz
O
H
O
NH N OH
HN
2OH FeCl
substrate
3 Fe III
O
substrate O
3
hydroxamic acid
Figure 4.20 Hydroxylamine trapping of an activated intermediate.
already mentioned is the trapping of imine intermediates formed upon reaction
of active site lysine residues with carbonyl substrates with sodium borohydride.
Another common example is the trapping of activated carbonyl intermediates
with hydroxylamine, a potent nitrogen nucleophile. This reaction forms hydroxamic
acid products which can be detected spectrophotometrically by treatment
with iron(III) chloride solutions, as shown in Figure 4.20.
Chemical inference
The existence of certain intermediates can be inferred by following the fate of
individual atoms in the substrate. For example, the existence of an acyl phosphate
intermediate in the glutamine synthetase reaction was established by
incubating 18 O-labelled substrate with enzyme and cofactor adenosine triphosphate
(ATP). The inorganic phosphate product was found to contain one atom
of 18 O, consistent with formation of the acyl phosphate intermediate as shown
in Figure 4.21.