Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Enzymatic Addition/Elimination Reactions 195
− O 2 C
H S
H R
HO
OH
OH
O
3-dehydroquinate
dehydratase
− O 2 C
Figure 8.3 Reaction catalysed by 3-dehydroquinate dehydratase.
OH
OH
O
+ H 2 O
b-hydroxy-ketone. This reaction is catalysed by 3-dehydroquinate dehydratase,
the third enzyme on the shikimate pathway.
The enzymatic reaction is reversible, although the equilibrium constant of
16 lies in favour of the forward reaction. The stereochemical course of the
enzymatic reaction in Escherichia coli was shown, using stereospeciWcally
labelled substrates, to be a syn-elimination of the equatorial C-2 proR hydrogen
and the C-1 hydroxyl group. The stereochemistry of the reaction is remarkable,
since the axial C-2 proS hydrogen is much more acidic in basic solution than the
proR hydrogen, due to favourable overlap with the C2O p bond.
A major clue to the enzyme mechanism is that the enzyme is inactivated
irreversibly by treatment with substrate and sodium borohydride. This indicates
that an imine linkage is formed between the C-3 ketone and the e-amino
group of an active site lysine residue. Peptide mapping studies have established
that this residue is Lys-170.
A mechanism for enzymatic reaction is shown in Figure 8.4. Upon formation
of the imine linkage at C-3, a conformational change is thought to take
place, giving a twist boat structure. In this conformation the C2H bond of the
proR hydrogen lies parallel to the orbital axes of the adjacent C2N p bond,
favouring removal of this hydrogen. Proton abstraction by an active site base
− O 2 C
*H
HO
OH
OH
O
Enz-NH 2
twist-boat conformation
− O 2 C
HO
EnzB −
H*
OH
OH
NHEnz
− O 2 C
HO
H*
EnzB
OH
OH
NHEnz
− O 2 C
OH
OH
O
− O 2 C
OH
OH
NHEnz
H
O
H
EnzB −
Figure 8.4 Mechanism for reaction catalysed by Escherichia coli 3-dehydroquinate dehydratase.