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If the pyridoxal-phosphate-binding lysine residue 258 of aspartate
aminotransferase is exchanged for a histidine residue, the enzyme retains
partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P.
(1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the
mutant enzymes of both chicken mitochondria and Escherichia coli were determined
at high resolution. The folding patterns of the polypeptide chains proved to be
identical to those of the wild-type enzymes, small conformational differences
being restricted to parts of the active site. If aspartate or glutamate was
added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm
(weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external
aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently
accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda
max 330 nm, positive CD), a putative ketamine intermediate could be detected;
however, with oxalacetate, an equilibrium between external aldimine and the
pyridoxal form, which was strongly in favour of the former, was established
within seconds. The transamination cycle with glutamate and oxalacetate proceeds
only three orders of magnitude more slowly than the overall reaction of the
wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25
degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant
apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H
with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the
wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to
some extent substitute for the active-site lysine residue. The imidazole ring of
H258, however, seems too distant from C alpha and C4' to act efficiently as
proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that
the prototropic shift might be mediated by an intervening water molecule.
Transmination of the internal to the external aldimine apparently can be
replaced by de novo formation of the latter, and by its hydrolysis in the
reverse direction.
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