Abstract |
Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism and subject to
complex allosteric regulation, exists in humans in two isoforms: the housekeeping hGDH1,
encoded by the GLUD1 gene, and hGDH2, which is expressed mainly in brain and testis and
encoded by GLUD2, a gene that emerged through retroposition ~20 million years ago.
Despite 97% sequence homology, with only 15 aminoacid differences between hGDH1 and
hGDH2, the two isoenzymes differ markedly in their properties: hGDH2 displays a
substantially lower basal activity in absence of activators, increased resistance to GTP
inhibition, and decreased thermostability.
In this work we explore structure-function relationships of the two isoenzymes through
mutagenesis and functional studies on wild-type and mutant hGDHs. We show that basal
activity of hGDH2 in vitro is dependent on enzyme concentration, temperature and buffer
composition. Furthermore, we show that the aminoacid differences R443S and G456A in the
allosteric region of the enzyme, shown previously to diminish basal activity and to increase
GTP-resistance, respectively, cannot reproduce the unique properties of hGDH2: the hGDH1-
R443S/G456A double mutant has much lower basal activity and heat resistance than hGDH2,
and is not activated by leucine. A chimeric enzyme, featuring all 4 aminoacid differences in
the allosteric region (hGDH1-M415L/R443S/G456A/R470H), can also not reproduce the
properties of hGDH2. The S174N aminoacid change, however, introduced into the
R443S/G456A double mutant, makes the resulting enzyme sensitive to leucine activation and
increases its basal activity and heat stability. Thus, aminoacid changes outside the allosteric
region are crucial for the unique properties of hGDH2. In addition, we show that basal
activity, heat stability and resistance to inhibition by estrogens are directly related, interlinked
to each other properties of hGDHs.
Finally, we show that a polymorphism in hGDH2 (S445A), that has been associated to
accelerated onset of Parkinson’s disease, probably exerts its effect by increasing hGDH2’s
basal activity and does not affect GTP inhibition. Mutations in the same region of hGDH1
(Q441R, S445L, S448P, K450E, H454Y), which cause the hyperinsulinism-hyperammonemia
syndrome through increasing hGDH1’s resistance to GTP, do not have a similar effect in
GTP-regulation when introduced to hGDH2, but can instead cause alterations in basal
activity, depending on their exact location.
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