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Enzymatic Carbon–Carbon Bond Formation 177
The Wrst step in each of the TPP-dependent reactions is deprotonation of the
thiazolium ring to generate a carbanion ylid at C-2. In chemical models the pK a
of this proton is 17–19, yet when bound to the enzyme it is able to exchange
with 2 H 2 O, therefore the enzyme active site increases the acidity of this position
dramatically. When bound to the active site, the TPP cofactor adopts a ‘V’
conformation (shown for pyruvate decarboxylase in Figure 7.26), which brings
N-4 0 of the aminopyrimidine ring in close proximity to C-2 of the thiazolium
ring. It is thought therefore that N-4 0 of an imine tautomer of the aminopyrimidine
ring deprotonates C-2, to generate the C-2 carbanion.
The ylid carbanion then attacks the keto group of pyruvate to generate a
covalently attached lactyl adduct (see Figure 7.27). Decarboxylation of the
lactyl adduct then takes place, using the carbon–nitrogen double bond as an
electron sink, forming an enamine intermediate. Protonation of this intermediate
and fragmentation of the linkage with the coenzyme yields acetaldehyde and
regenerates the coenzyme ylid. Alternatively, the enamine intermediate can
react with an electrophile (E þ ), generating after fragmentation of the linkage
with the coenzyme, an acetyl-E species.
An important reaction in the cellular breakdown of d-glucose is the
conversion of pyruvate to acetyl CoA, which is catalysed by the pyruvate
Figure 7.26 Active site of yeast pyruvate decarboxylase (PDB Wle 1QPB), showing the ‘V’ conformation
of the bound TPP cofactor, which is shown in black. Three active site residues, Asp-28, Glu-51
and Ile-415, are shown in red.