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Enzymatic Transformations of Amino Acids 215
HO
HO
H
CO 2 H
NH 3
L-dopa
decarboxylase
PLP
HO
HO
dopamine
NH 3
+ CO 2
+ CO 2
HO
N
H
H
NH 3
CO 2 H
L-dopa
decarboxylase
PLP
Figure 9.6 Reactions catalysed by l-dopa decarboxylase.
HO
N
H
serotonin
NH 3
9.3 CASE STUDY: Aspartate aminotransferase
Aspartate aminotransferase catalyses the transamination of l-aspartic acid into
oxaloacetate, at the same time converting a-ketoglutarate into l-glutamic acid.
The enzymatic reaction, therefore, consists of two half-reactions, shown in
Figure 9.7.
Mammals contain two forms of aspartate aminotransferase, a cytosolic
form and a mitochondrial form, both of which have been puriWed and studied
extensively. The bacterial enzyme from Escherichia coli has also been puriWed,
overexpressed and crystallised, allowing a detailed study of its mechanism of
action, which is depicted in Figure 9.8. The resting state of the enzyme in the
absence of substrate contains the Lys-258–PLP–aldimine adduct, which
absorbs at 430 nm. Upon binding of l-aspartate, the PLP–aldimine intermediate
is formed, which also absorbs at 430 nm. The e-amino group of Lys-258,
released from binding the PLP cofactor, acts as a base for deprotonation of the
a-hydrogen. This forms the quinonoid intermediate (visible at 490 nm by
stopped Xow kinetics) also found in the PLP-dependent racemases. However,
in this case the quinonoid intermediate is reprotonated adjacent to the heterocyclic
ring, generating a ketimine intermediate which can be observed at 340 nm
by stopped Xow kinetics. Hydrolysis of the ketimine intermediate releases the
product oxaloacetate, and generates a modiWed form of the cofactor known as
pyridoxamine 5 0 -phosphate (PMP), visible at 330 nm.
− O 2 C
H
CO −
2
aspartate
aminotransferase
− O 2 C
CO 2
−
NH 3
O
− O 2 C
H
CO −
2
PLP
PMP
− O 2 C
CO 2
−
NH 3
Figure 9.7 Two half-reactions catalysed by aspartate aminotransferase.
O