Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

64 Chapter 4
In general the stereochemical course of an enzymatic reaction is
usually determined by replacement of particular atoms of the substrate by
isotopes of carbon, hydrogen, oxygen and nitrogen, whose fate at the end
of the reaction can then be monitored. In particular, enzymatic reactions
involving prochiral centres can only be studied by replacement of one of the
prochiral substituents by another isotope, thus generating a chiral centre.
The isotopes available are listed in Table 4.2, together with the methods
available for their analysis. In some cases such as 1 H, 13 C and 15 N these nuclei
possess nuclear spin, which allows analysis by NMR spectroscopy. In other
cases, such as 3 H and 14 C, the nuclei are radioactive and their presence can
be detected by scintillation counting. Isotopes such as 18 O which are
neither radioactive nor possess nuclear spin must be detected either by mass
spectrometry or by their eVect on neighbouring nuclei: in the case of 18 O their
attachment to a neighbouring 13 C shifts the 13 C NMR signal upWeld by
0.01–0.05 ppm.
Enzymatic reactions which involve the substitution of one group for
another group in a deWned relative orientation can either take place with
retention or inversion of stereochemistry. Where such reactions occur at a
prochiral centre, the stereochemical course can be examined by synthesis of
a stereospeciWcally labelled substrate. For example, the haem enzyme P450cam
Table 4.2 Isotopes available for stereochemical elucidation.
Isotope
Natural
abundance (%)
Method of analysis
Hydrogen 1 H 99.985 NMR (I ¼ 1/2)
2 H 0.015 NMR (I ¼ 1) or shift in 13 C NMR
3 H — Scintillation counting
Carbon 12 C 98.9
13 C 1.1 NMR (I ¼ 1/2)
14 C — Scintillation counting
Nitrogen 14 N 99.63
15 N 0.37 NMR (I ¼ 1/2)
Oxygen 16 O 99.8
17 O 0.037 NMR (I ¼ 5/2)
18 O 0.20 Mass spectrometry or shift in 13 C NMR