Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

70 Chapter 4
18O 18O 18 O
O
P O −
18O
NH 3
+ 18 O −
ENZ
O −
+ ATP ADP
H 3 N CO −
2
+
H 3 N CO −
2
+
H 3 N CO −
2
O
P
O −
O −
Figure 4.21 IdentiWcation of glutamine synthetase intermediate by chemical inference. ADP,
adenasine diphosphate; NH 3 , ammonia.
Isotope exchange
If an enzyme reaction involves the reaction of two species to form an intermediate
which is then attacked by a third species, the Wrst step or partial reaction can
be analysed in isolation using isotope exchange. Taking the glutamine synthetase
reaction as an example, glutamate Wrst reacts with ATP to form adenosine
diphosphate (ADP) and an intermediate g-glutamyl phosphate, which is
attacked by ammonia to form glutamine. If the enzyme is incubated with
glutamate, 14 C-ATP and unlabelled ADP in the absence of ammonia, then
the enzyme cannot complete the overall reaction, but it can convert glutamate
to enzyme-bound g-glutamyl phosphate and 14 C-ADP. In this case 14 C-ADP
can be released from the enzyme without releasing the intermediate. The
enzyme can then convert unlabelled ADP to ATP via the reverse reaction.
Overall this process leads to the ‘exchange’ of 14 C label from ATP to ADP, as
illustrated in Figure 4.22.
This method depends on the ability of the enzyme to release 14 C-ADP at the
intermediate stage. Since many enzymes have well-deWned orders of binding of
their substrates, this release may be slow or even impossible in the absence of
the third substrate. A more subtle method of analysing such exchange is the
method of positional isotope exchange developed by Rose. This method is
illustrated in Figure 4.23 for the glutamine synthetase reaction.
For this method ATP is labelled with 18 O at the b,g-O bridge position.
Upon formation of the reaction intermediate the g-phosphate is transferred
to glutamate, allowing the 18 O label to scramble amongst the b-phosphate
oxygens whilst still bound to the enzyme. Upon reformation of the b,g-O
bridge in the reverse reaction the 18 O label will be present not only in the bridge
14 C-ATP 14 C-ADP
L-Glu
L-Glu-γ-PO 3
2−
NH 3
L-Gln
ATP ADP
Figure 4.22 Isotope exchange in the glutamine synthetase reaction. l-Gln, glutamine; l-Glu,
glutamate; l-Glu--PO 2 3 , -glutamyl phosphate.