Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

196 Chapter 8
leads to the formation of a planar enamine intermediate. This intermediate acts
as a stabilised carbanion intermediate for an E1cb elimination mechanism.
Extrusion of water, presumably assisted by general acid catalysis, is followed
by hydrolysis of the iminium salt linkage to give 3-dehydroshikimic acid.
The elimination of b-hydroxyacyl coenzyme A (CoA) thioesters is one of
the reactions involved in the fatty acid synthase cycle described in Section 7.4.
b-Hydroxydecanoyl thioester dehydratase catalyses the reversible dehydration
of b-hydroxydecanoyl thioesters to give trans-2-decenoyl thioesters, which are
subsequently isomerised to give cis-3-decenoyl thioesters. This reaction
sequence represents a branch point in the biosynthesis of saturated and unsaturated
fatty acids, as shown in Figure 8.5.
b-Hydroxydecanoyl thioester dehydratase from E. coli is a dimer of
sub-unit molecular weight 18 kDa and requires no cofactors for activity.
Stereochemical studies have revealed that the proS hydrogen is abstracted at
C-2, so elimination of the 3R alcohol results in an overall syn-elimination of
water. The subsequent isomerisation involves abstraction of the proR hydrogen
at C-4. There is considerable evidence for a histidine active site base, which has
been identiWed by peptide mapping studies as His-70. It is thought that His-70
also mediates the isomerisation reaction, as shown in Figure 8.6. However,
there is no intramolecular transfer of deuterium from C-4 to C-2, suggesting
that the protonated His-70 exchanges readily with solvent water.
This enzyme is speciWcally inactivated by a substrate analogue 3-decynoyl-N-acetyl-cysteamine.
The a-proton of the inhibitor is abstracted by
His-70 in the normal fashion, but protonation of the alkyne at the g-position
generates a highly reactive allene intermediate. This intermediate is then
attacked by the nearby His-70 resulting in irreversible inactivation, as shown
in Figure 8.7. Since the inhibitor is activated by deprotonation at C-2 the
enzyme brings about its own inactivation by way of the enzyme mechanism.
This class of mechanism-based inhibitors are, therefore, often known as ‘suicide
inhibitors’.
The dehydration of b-hydroxy-carboxylic acids is a reaction which occurs
quite frequently in biochemical pathways. Two dehydrations which occur on
the citric acid cycle are shown in Figure 8.8: the dehydration of citrate and
rehydration of cis-aconitate to give isocitrate, catalysed by the single enzyme
OH
O
SR
dehydratase
O
SR
saturated
fatty acids
isomerase
O
SR
Figure 8.5 Reactions catalysed by b-hydroxydecanoyl thioester dehydratase.
unsaturated
fatty acids