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202 Chapter 8
EnzB −
2 H
− O 2 C
Me
H
CO −
2
methylaspartase
2 H H
CO −
2 15 NH +
3 − O 2 C
Me 15 NH +
2
− O 2 C
Me
H
+
− CO 2
15 NH 3
Enz
Figure 8.14 Concerted C2H and C2N cleavage in the methylaspartase reaction.
One would expect to see a primary kinetic isotope eVect for b- 2 H-labelled
substrates, which is not observed for phenylalanine ammonia lyase, although a
kinetic isotope eVect is observed for histidine ammonia lyase. An alternative
mechanism (path B) involving C2C bond formation with the aryl ring of
phenylalanine has recently been proposed for phenylalanine ammonia lyase,
analogous to a Friedel–Crafts reaction.
An important mechanistic question is whether the elimination of the substrate
is concerted or stepwise This question has been addressed using kinetic
isotope eVects in the reaction of methylaspartase, shown in Figure 8.14. This
enzyme catalyses the anti-elimination of threo-b-methyl-aspartic acid, also
utilising a dehydroalanine cofactor. Measurement of the rate of the enzymatic
reaction using the [3- 2 H] substrate revealed a primary kinetic isotope eVect of
1.7, indicating that C2H bond cleavage is partially rate-determining. However,
there was also found to be a 15 N isotope eVect of 1.025 upon the [2- 15 N] labelled
substrate. If the reaction is concerted, then both of these kinetic isotope eVects
are operating on the same step, in which case the eVects should be additive. So a
[3- 2 H, 2- 15 N] substrate was prepared and a kinetic isotope eVect of 1.042 was
observed, indicating that the isotope eVects are additive and that the elimination
is indeed concerted.
Interestingly, the enzyme aspartase, which catalyses the elimination of aspartic
acid, does not contain the methylene-imidazolone cofactor, thus Nature is
able to catalyse the elimination of ammonia without the assistance of covalent
catalysis.
8.4 Elimination of phosphate and pyrophosphate
In all elimination reactions an important determinant of reaction rate and
mechanism is whether a good leaving group is available. Elimination of water
is hindered by the fact that the hydroxyl group is a poor leaving group, since the
pK a of the conjugate acid water is 15.7. In the dehydratase enzymes this
problem is alleviated by acid or Lewis acid catalysis. However, another strategy
found in biochemical pathways for provision of an eYcient leaving group is
phosphorylation of the leaving group.
The pK a values for the three dissociation equilibria of phosphoric acid, as
shown in Figure 8.15, are 2.1, 7.2 and 12.3. At neutral pH a phosphate mono-