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Methods for Studying Enzymatic Reactions 67
monitoring the fate of the 3 H, either to water or to tritiated fumaric acid.
Since the stereochemistry of the fumarase and malate synthase reactions is
known, the conWguration of the chiral methyl group can be deduced. By this
method a number of such methylene-to-methyl interconversions have been
analysed.
A similar stereochemical strategy is used to analyse the stereochemistry
of phosphoryl transfer reactions, since phosphates also contain three apparently
identical oxygen substituents. Three isotopes of oxygen are also available:
16 O, 17 O and 18 O. Using skilful synthetic chemistry approaches, phosphate
ester substrates can be prepared containing all three isotopes of oxygen. Incubation
of the chiral phosphate ester substrate with the corresponding phosphotransferase
enzyme generates a chiral phosphate ester product, as shown in
Figure 4.16. The conWguration of the chiral product reveals whether the enzymatic
reaction proceeds with retention or inversion of conWguration. Analysis of
the conWguration of the chiral phosphate ester product is complicated, but in
essence involves chemical or enzymatic conversion to a diastereomeric derivative,
followed by 31 P NMR spectroscopic analysis.
The stereochemistry of reactions releasing inorganic phosphate presents
an even more diYcult problem, since there are four apparently identical
oxygens to be distinguished, but only three isotopes of oxygen. This has been
solved by incorporating one atom of sulphur as a substituent, since thiophosphate
ester substrates are accepted by these enzymes (Figure 4.17). Again the
conWguration of the [ 16 O, 17 O, 18 O]-thiophosphate product can be deduced by
conversion to a diastereomeric derivative followed by NMR spectroscopic
analysis. For a detailed discussion of this stereochemical analysis the interested
reader is referred to the further reading at the end of the chapter.
R
O
16 O
16 O
P
18 O − + R'O − ENZ
18
− O P O R'
17 O − 17 O −
convert to
diastereomeric
derivative
31 P NMR
INVERSION
Figure 4.16 Stereochemistry of phosphoryl transfer reaction.
S
S
ENZ
R O P
P
H 16 −
2 O
O
18 O − 18 O −
17 O − 16
17 O −
RETENTION
Figure 4.17 Stereochemistry of phosphate release.