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J. Biol. Chem., Vol. 277, Issue 48, 46552-46558, November 29, 2002

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Substitution of Ser for Arg-443 in the Regulatory Domain of Human Housekeeping (GLUD1) Glutamate Dehydrogenase Virtually Abolishes Basal Activity and Markedly Alters the Activation of the Enzyme by ADP and L-Leucine^*

Ioannis Zaganas, Cleanthe Spanaki, Michael Karpusas^§, and Andreas Plaitakis^¶

From the Departments of  Neurology and ^§ Basic Sciences, University of Crete, School of Health Sciences, Section of Medicine, 71500 Heraklion, Crete, Greece

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by L-leucine (1-10 mM) in the absence of other effectors. However, when ADP (0.025-0.1 mM) was present in the reaction mixture, L-leucine (0.3-6.0 mM) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC₅₀ = 383.9 ± 14.6 µM) than the GLUD1 GDH (SC₅₀ = 31.7 ± 4.2 µM; p < 0.001); however, at 1 mM ADP the V_max for the mutant (136.67 µmol min¹ mg¹) was comparable with that of the GLUD1 GDH (152.95 µmol min¹ mg¹). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.

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