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Enzymatic Carbon–Carbon Bond Formation 159
The reaction catalysed by the class I enzyme from rabbit muscle has been
shown to proceed via formation of an imine linkage between the e-amino group
of an active site lysine residue and the C-2 carbonyl of DHAP. The imine
linkage can be reduced by sodium borohydride to give an irreversibly inactivated
secondary amine. Incubation of enzyme with radiolabelled G3P and
sodium borohydride followed by proteolytic digestion of the inactivated
enzyme has identiWed the imine-forming active site residue as Lys-229.
Incubation of enzyme and DHAP in 2 H 2 O (in the absence of G3P) leads to
stereospeciWc 2 H exchange at C-3 to give 3S-[3- 2 H]-DHAP. This indicates that
the proS proton at C-3 is speciWcally removed following imine formation, giving
an enamine intermediate. In order to generate the observed stereochemistry at C-
3 and C-4 of the product the enamine intermediate must react from its si-face
with the si-face of the aldehyde group of G3P (see Figure 7.5 later). The resulting
imine intermediate is hydrolysed to yield the product fructose-1,6-bisphosphate.
An X-ray crystal structure was determined for rabbit muscle fructose-
1,6-bisphosphate aldolase in 1987. The tertiary structure of the protein subunit
is an ab barrel consisting of 9 parallel b-sheets interconnected by a-helices.
The active site lies in the centre of the barrel formed by the b-sheets, as shown in
Figure 7.4. Close to the imine-forming Lys-229 in the centre of the active site
cavity are three active site residues which participate in catalysis: Glu-187,
Lys-146 and Asp-33. Site-directed mutagenesis has been used to replace
each of these residues with a non-functional side chain. Replacement of
Glu-187 by alanine dramatically lowers the rate of SchiV base formation,
indicating that Glu-187 participates in acid–base catalysis during SchiV base
formation. Replacement of Asp-33 by alanine dramatically reduces the rate of
C2C bond formation, consistent with a role as a catalytic base in C2C bond
cleavage. Mutation of Lys-146 to alanine gives a mutant enzyme which is
> 10 6 -fold less active, whilst a histidine mutant enzyme is only 2000-fold less
active than the wild type enzyme, suggesting that Lys-146 fulWls an acid–base
function.
The most plausible catalytic mechanism is shown in Figure 7.5. SchiV base
formation with Lys-229 is assisted by nearby Glu-187 and Lys-146. Asp-33 acts
as the base for enamine formation, and protonates the carbonyl oxygen of G3P
on the same face.
The fructose-1,6-bisphosphate aldolase enzyme in yeast and bacteria is
a class II enzyme, dependent upon Zn 2þ for activity. Studies of the yeast
aldolase by infrared spectroscopy have shown that the carbonyl stretching
frequency of G3P is shifted from 1730 cm 1 in aqueous solution to 1706 cm 1
when bound to the enzyme, whereas the stretching frequency of the DHAP is
unchanged upon binding. These data indicate that the aldehyde of G3P is
polarised when bound to the active site. The X-ray crystal structure of the
Escherichia coli aldolase has been solved, revealing that the ketone and hydroxyl
groups of DHAP are chelated to the Zn 2þ cofactor, which is ligated by