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Enzymatic Carbon–Carbon Bond Formation 165
H 3 C
O
SCoA
+
H 3 C
O
SCoA
β-ketoacyl
thiolase
O
O
SCoA
+ CoASH
O
hydroxymethylglutaryl
CoA synthase
OH
− O 2 C
OH
mevalonic acid
HMG CoA
reductase
2 NADPH
CoAS CH 3
O
− O 2 C
OH
SCoA
O
O OH
+
− O 2 C
CoAS CH
CO −
2
3 R CO −
2 R
malate synthase R = H
citrate synthase R = CH 2 CO 2 H
Figure 7.11 Reactions catalysed by Claisen enzymes, involving acetyl CoA.
Mevalonic acid is an important cellular precursor to terpene and steroid
natural products. The biosynthesis of mevalonic acid involves two reactions in
which carbon–carbon bonds are formed from the a-position of acetyl CoA. The
Wrst reaction is a condensation reaction with a second molecule of acetyl CoA
to form 3-ketobutyryl CoA. Note that the good leaving group properties of the
thiol group of CoA are also signiWcant in this reaction. Reaction of the b-keto
group with a further equivalent of acetyl CoA generates hydroxymethylglutaryl
CoA, which is reduced by a nicotinamide adenine dinucleotide
(NADPH)-dependent reductase to give mevalonic acid. Two further examples
shown in Figure 7.11 are malate synthase and citrate synthase, which catalyse
important metabolic reactions of acetyl CoA.
The mechanism of these ‘Claisen enzymes’ which react through the
a-position of acetyl CoA could either proceed via formation of an a-carbanion
intermediate, or by a concerted deprotonation/bond formation step. Incubation
of acetyl CoA with malate synthase, or citrate synthase in the presence of 3 H 2 O
followed by re-isolation of substrate, gives no exchange of 3 H into acetyl CoA,
ruling out the reversible formation of a carbanion intermediate. Stereochemical
studies using chiral [2- 2 H, 3 H]-labelled acetyl CoA illustrated in Figure 7.12,
have established that the malate synthase reaction proceeds with overall inversion
of stereochemistry, and with a kinetic isotope eVect (k H =k T ¼ 2:7).
These results suggest that there is a transient thioester enolate intermediate
formed in the malate synthase reaction, which then reacts with the aldehyde
carbonyl of glyoxylate. Formation of the thioester enolate intermediate would
be thermodynamically unfavourable due to the high pK a of the a-proton;