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Enzymatic Carbon–Carbon Bond Formation 171
O
CO 2 H
+ CO 2
pyruvate carboxylase
biotin, ATP
HO 2 C
O
CO 2 H
O
SCoA
+ CO 2
acetyl CoA carboxylase
biotin, ATP
HO 2 C
O
SCoA
O
1 3
HN NH
H H
S
biotin
O
H
N
HO
Lys-Enz
O
N
H
O
S
NH
H
N 1 -carboxy-biotin
H
N Lys-Enz
O
Figure 7.17 Biotin-dependent enzymes.
species N 1 -carboxy-biotin, illustrated in Figure 7.17, has been shown to be chemically
and kinetically competent as an intermediate in the carboxylation reaction.
Incubation of enzyme with 18 O-labelled bicarbonate led to the isolation of
product containing two atoms of 18 O and inorganic phosphate containing one
atom of 18 O. The transfer of 18 O to inorganic phosphate implies that ATP is used
to activate bicarbonate by formation of an acyl phosphate intermediate, known
as carboxyphosphate. It is thought that carboxyphosphate is then attacked by
N-1 of the biotin cofactor. Since the NH group of amides and urea is normally a
very unreactive nucleophile, it is thought that N-1 is deprotonated prior to
attack on carboxyphosphate. The carboxy-biotin intermediate thus formed is
then attacked by a deprotonated substrate, probably by initial decarboxylation
to generate carbon dioxide, to form the carboxylated product and regenerate the
biotin cofactor, as shown in Figure 7.18. It has been shown that the carboxylation
of pyruvate to oxaloacetate catalysed by pyruvate carboxylase proceeds
with retention of conWguration at C-3, as shown in Figure 7.19. Other biotindependent
carboxylases also proceed with retention of conWguration.
7.6 Ribulose bisphosphate carboxylase/oxygenase (Rubisco)
All of the carbon-based moleules found in living systems are ultimately derived
from the Wxation of gaseous carbon dioxide by green plants during photosynthesis.
Carbon dioxide is then regenerated from respiration of living organisms,
from the decomposition of carbon-based material and dead organisms, and
from the combustion of wood and fossil fuels. This global cycle of processes is
known as the carbon cycle. The enzyme which is responsible for the Wxation of
carbon dioxide by green plants is an enzyme of primary importance to life on
earth. This enzyme is ribulose bisphosphate carboxylate/oxygenase, commonly
known as Rubisco.