Abstract |
Glutamate dehydrogenase (GDH), an enzyme central to glutamate
metabolism, is located in the mitochondria. In addition, there is evidence for extramitochondrial
localization of GDH. Human GDH exists in housekeeping (hGDH1)
and nerve tissue-specific (hGDH2) isoenzymes encoded by the GLUD1 and the
GLUD2 genes respectively, which differ markedly in their basal activity, allosteric
regulation and thermal stability. hGDH2 is thermolabile, shows low basal activity that
is fully restored by ADP albeit at higher concentrations than hGDH1, is more
sensitive to L-leucine activation and to the synergistic effect of ADP and L-leucine
and resistant to GTP inhibition. Site directed mutagenesis of GLUD1 revealed that
replacement of Gly456 by Ala made the enzyme resistant to GTP without altering its
regulation by ADP. In addition substitution of Ser for Arg443 (which lies in the
antenna region of GDH) virtually abolished basal activity, made the enzyme
extremely sensitive to heat inactivation and totally abrogated the activation of the
enzyme by L-Leucine. However the presence of low concentrations of ADP (0.025-
0.1 mM) permitted the activation of the mutant by L-leucine. Structural modeling
implicated that the replacement of Arg443 by Ser may disrupt the H-bond(s) between
Arg443 and Ser409 (part of the ascending strand of the antenna) from an adjacent
subunit and thus may result in closure of the active catalytic cleft. Substitution of the
Ser409 for Arg rendered the enzyme virtually inactive at baseline and resistant to Lleucine
activation. ADP fully restored the activity of mutant and made it sensitive to
L-leucine activation. On the other hand, replacement of Ser409 by Asp had a lesser
effect on basal activity and to L-leucine activation than Arg substitution. Hence, these
data confirm that interaction of adjacent subunits through the Arg443 and Ser409
residues plays a crucial role in setting the basal activity levels. Abrogation of Lleucine
activation by R443S, S409R or S409D mutants is consistent with the concept
that these mutations lead to a closed conformation, which hinders access of L-leucine
to the catalytic site for it action. Since the S409R hGDH1 mutant was insensitive to
heat denaturation, the interaction between the two residues does not seem to be
responsible for heat sensitivity. In addition both S409R and S409D hGDH1 mutants
showed a differential response to GTP inhibition, with the former being more
sensitive and the latter more resistant to GTP in comparison to the wt hGDH1.
Moreover, we created a double hGDH1 mutant that had both Arg443Ser and
Gly456Ala in the same polypeptide chain. Functional analyses revealed that the
doubly mutated enzyme had similar properties but did not acquire all the
characteristics of the wild-type hGDH2. Subcellular localization studies were
performed using expression vectors for hGDH1 and hGDH2 fused with the enhanced
green fluorescence protein (EGFP) that transiently transfected COS 7, HeLa, CHO,
HEK 293 and neuroblastoma cell lines. When the cultured cells were transfected with
EGFP-tagged hGDH1 or hGDH2, confocal microscopy demonstrated localization of
the fluorescence in the cytoplasmic region within coarse structures resembling
mitochondria. Co-transfection experiments using a mixture of hGDH1-EGFP (or
hGDH2-EGFP) vector and the pDsRed2-Mito vector (a mitochondrial marker)
showed an identical fluorescence pattern in merged pictures, thus confirming the
mitochondrial localization of both human GDHs. In addition, a small fraction of the
hGDH1 (or hGDH2) was found to co-localize with endoplasmic reticular marker
pDsRed2-ER. There was no evidence for the nuclear or cytoplasmic localization of
either GDH isoforms. Our results elucidate further the properties that allow the brain
isoenzyme to function well under the special conditions prevailing in the human
Central Nervous System.
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