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228 Chapter 10
B 1
–
H
+
NH 3
CO −
2
H B 2
B 1
H
+
H 3 N CO −
2
H B 2
R
R
B 1
− O 2 C
H 3 N
H +
R
H – B 2
Figure 10.2 Two-base mechanism for cofactor-independent racemases.
cus thermophilus. There is evidence in both these enzyme-catalysed reactions for
a two-base mechanism of racemisation, as shown in Figure 10.2. In this mechanism
an a-proton is removed from one face of the amino acid by an active
site base and a proton delivered onto the other face by a protonated base on
the opposite side of the enzyme active site. In this mechanism the unstable
a-carbanion would exist only Xeetingly.
How is experimental evidence obtained for such a two-base mechanism
One such method is illustrated in Figure 10.3. In this experiment a stoichiometric
amount of enzyme is incubated with substrate for a short period of time in
tritiated water, and the distribution of 3 H label examined in the products. In a
one-base active site the a-hydrogen is removed from the l-enantiomer and
returned to the opposite face by the same base, giving the d-enantiomer.
Hence any 3 H label incorporated by exchange of the active site base with
3 H 2 O would be delivered equally to both l- andd-enantiomers. However, in
a single catalytic cycle of a two-base active site one would expect the a-proton
of the l-enantiomer to be abstracted by one base and a 3 H label to be delivered
from the opposite face by the other base, resulting in increased incorporation of
3 H label into the d-enantiomer. Thus, starting with l-glutamate, glutamate
racemase catalyses the preferential incorporation of 3 H from 3 H 2 O into
d-glutamic acid.
This approach has established the likelihood of a two-base mechanism for a
number of cofactor-independent racemases and epimerases. However, this does
not explain how the intermediate carbanion, however Xeeting, is stabilised.
B 1
–
H
CO −
2
+
NH 3 3 H B 2
glutamate
racemase
3 H 2 O
single turnover
B 1
− O 2 C
H
3 H – B 2
H 3 N +
CO 2
−
− O 2 C
1 H-L-glutamic acid 3 H-D-glutamic acid
Figure 10.3 Experimental evidence in favour of a two-base mechanism.