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Methods for Studying Enzymatic Reactions 65
O
2 H
P450cam
O 2 , NADPH
O
OH
1 H
1 H
RETENTION
Figure 4.14 Stereochemistry of P450cam-catalysed reaction.
which catalyses the hydroxylation of camphor was shown to proceed with
retention of stereochemistry by use of the labelled substrate illustrated in
Figure 4.14.
Interconversions of methylene (5CH 2 or 2CH 2 2) groups to methyl
(2CH 3 ) groups require a special type of stereochemical analysis, since the
resulting methyl group contains three apparently identical hydrogen atoms
(i.e. an XXXY system). However, it is possible to analyse these methyleneto-methyl
interconversions using all three of the isotopes of hydrogen – 1 H, 2 H
and 3 H – in the form of a chiral methyl group. It is important to note that in this
analysis the 1 Hand 2 H substituents are present in 100% abundance, whereas
only a small proportion of molecules contain 3 H (since 3 H is only available and
only safe to handle in relatively low abundance). Therefore the analysis of
chiral methyl groups must focus on those molecules containing 3 H, by detecting
the presence or absence of 3 H label.
Chiral methyl groups can be generated from enzymatic reactions by
preparing the methylene substrate in a stereospeciWcally labelled form using
two of the isotopes of hydrogen, and carrying out the enzymatic reaction in
the presence of the third isotope. If the product can be degraded to chiral
acetic acid, then the conWguration of the chiral methyl group can be determined
using a method developed independently by Cornforth and Arigoni, shown in
Figure 4.15.
The method of analysis involves conversion to chiral acetyl coenzyme
A (CoA) (see Section 5.4), followed by incubation with malate synthase
(see Section 7.3), which removes one of the hydrogens on the methyl group,
and combines with glyoxalate to form malic acid. The malate synthase reaction
has a preference for removal of 1 H rather than 2 Hor 3 H (i.e. a kinetic isotope
eVect of k H =k T ¼ 2:7), so in the majority of molecules 1 H is removed. The
reaction with glyoxalate then occurs with inversion of conWguration. Therefore,
the 2S enantiomer of acetyl CoA is converted into the 2S,3R enantiomer of
malate containing 2 H and 3 H stereospeciWcally at C-3, as illustrated in Figure
4.15. Treatment with the enzyme fumarase then results in a stereospeciWc antielimination
of water, the enzyme removing only the proR hydrogen at C-3. The
conWguration of the major product at C-3 can, therefore, be deduced by