Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

216 Chapter 9
Arg 292 Arg 292
Lys 258
− Arg 386
O 2 C
− Arg386
O 2 C
*H CO −
2 CO −
Lys258
2
NH 2 +
NH + NH 2 H*
NH +
2− O 3 PO
+
N
H
OH
2− O 3 PO
OH
N
430 nm H
490 nm
− O 2 C
CO 2
−
− O 2 C
2− O 3 PO
O
Lys 258
NH 2
H
NH 2
*H
H 2 O
OH
2− O 3 PO
CO −
2
NH +
OH
+
N
H
330 nm
+
N
H
340 nm
Figure 9.8 Mechanism for aspartate aminotransferase half-reaction.
The reaction is completed by carrying out the reverse transamination on the
other a-keto acid substrate for this enzyme. a-Ketoglutarate is bound via a
ketimine linkage, which is isomerised as before to the aldimine intermediate.
Displacement of the aldimine linkage by Lys-258 releases l-glutamic acid and
regenerates the PLP form of the cofactor.
Examination of the X-ray crystal structure of aspartate aminotransferase,
illustrated in Figure 9.9, reveals that Lys-258 is suitably positioned to act as an
intramolecular base for proton transfer. Replacement of Lys-258 for alanine by
site-directed mutagenesis gives a completely inactive mutant enzyme, as
expected, since there is no point of attachment or active site base. A Cys-258
mutant enzyme is similarly inactive. However, if this mutant is alkylated with
2-bromoethylamine an active enzyme is obtained which contains a thioether
analogue of lysine at its active site, as shown in Figure 9.10. This enzyme has
7% of the activity of the wild-type enzyme with a slightly shifted pH/rate proWle
of enzymatic activity, since the thioether-containing lysine analogue is slightly
less basic than lysine.
Examination of the active site of aspartate aminotransferase, shown in
Figure 9.9, reveals that both carboxylate groups of l-aspartate are bound by
electrostatic interactions to active site arginine residues: the a-carboxylate