610
Chirality in Biology
HOOC
H
H
COOH
OH
2
H
+
OH
2
H
COOH
H
H
HOOC
COOH
C
C
COOH
H
2
H(
R
)
H
OH(
S
)
2
3
CH
2
COOH
C
CH
2
COOH
HOOC
OH
OH
HOOC
CH
2
X
XH
2
C
pro-
R
pro-
S
O
CH
2
X
HOOC
H
+
CoA
S
OC
H
3
C
(a)
(b)
Fig. 22
The fumarase and citrate synthase reactions. (a) The top line shows
fumarase addition of
2
H
2
O to fumarate leading to production of
(2
R
,3
S
)-[2-
2
H]malate. (b) The lower line shows the addition of acetyl-CoA to
oxaloacetate; X
=
COOH. Use of
13
C-labeled acetyl-CoA (
13
Cident
iFedas
)
yields
13
Cinthepro-
S
position of citrate.
Similar considerations apply to addition
reactions at double bonds, C
=
C. Thus,
in the action of fumarase on fumarate in
2
H
2
O, the OH group attacks from below
the double-bond plane and H
+
enters from
above the plane, thus leading to formation
of (2
S
,3
R
)-[3-
2
H]-malic acid (Fig. 22).
Since
2
H is readily available at 100%
concentration,
2
H
CH
2
COOH can be
prepared. In this compound, the two
1
H atoms are in a prostereoisomeric re-
lationship. If a further substitution of
one
1
H by 100%
3
Hw
e
r
ep
r
a
c
t
i
c
a
b
l
e
,
a chiral compound would be formed,
[
3
H,
2
H,
1
H]-acetate, and theoretically op-
tical activity would be observable. Because
of the very high level of radioactivity as-
sociated with 100%
3
H, it is unlikely that
this statement will be put to the proof.
However, normal tracer levels of
3
Hcan
be used to explore the chirality of methyl
groups containing all three of the hy-
drogen isotopes. The two enantiomeric
acetates (Fig. 23a, b) can be synthesized ei-
ther chemically or by combined enzymatic
and
chemical
methods;
their
chirality
is demonstrated as follows. The acetate
samples are converted to the CoA deriva-
tives and the latter are used as sub-
strates in the malate synthase reaction
(EC 4.1.3.2; glyoxylate
+
acetyl-CoA
L-
(
S
)-malate). In this reaction, a marked
isotope effect dictates removal of
1
Hin
preference to either
2
Ho
r
3
H. In turn,
the malate samples are treated with fu-
marase (EC 4.2.1.2, fumarate hydratase,
L-(
S
)-malate
H
2
O
+
fumarate) and the
fumarate is examined for
3
H radioactivity
(Fig. 23).
The overall result is that from (
R
)-
[
1
H,
2
H,
3
H]-) acetate,
3
H is predominantly
retained in the isolated fumarate (ca. 80%
retention) and from the (
S
) enantiomer,
3
H is predominantly lost (ca. 20% reten-
tion). While loss/retention of
3
Hi
sno
t
100%, such completeness is not expected;
the actual value depends on the nature of
the isotope effect in the malate synthase
reaction. This malate synthase/fumarase
system demonstrates the chirality of the
methyl group and provides a convenient
assay method for the chirality of acetate
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