Abstract
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 (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). 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.
Highlights
Mammalian glutamate dehydrogenase (GDH)1 (E.C.1.4.1.3) catalyzes the reversible oxidative deamination of glutamate to ␣-ketoglutarate using NAD(H) or NADP(H) as co-factors (1)
Because the functional differences between the housekeeping and the nerve tissue-specific GDH arise from amino acid residues not common between the two isoenzymes, we have sought to identify these critical residues by mutagenizing the GLUD1 gene at sites that differ from the corresponding sites of the GLUD2 gene
Studies on Crude Tissue Extracts—Enzymatic assays carried out in crude tissue extracts showed that the S331T, M370L, and M415L mutants exhibited a basal catalytic activity similar to that of the wild-type GLUD1 GDH
Summary
Materials—Sf21 cells and the baculovirus expression vectors were obtained from Invitrogen. Cells of the insect Spodoptera frugiperda (Sf21) were co-transfected with the plasmid DNA (pVL1393 vector containing the GLUD1 insert) and modified baculovirus DNA (BaculoGold, BD Pharmingen), and incubated at 27 °C for 4 –5 days. The reaction mixture of 1 ml contained 50 mM triethanolamine (TRA) pH 8.0, buffer (except as indicated), 100 mM ammonium acetate, 100 M NADPH, 2.6 mM EDTA, and 0.05– 0.2 mg of crude extract protein or 0.3– 0.5 g of purified GDH, unless specified otherwise. Regulation of the human recombinant GDHs by ADP and/or L-leucine was studied essentially as described previously (15) by adding this compound to the reaction mixture at various concentrations (ADP ϭ 0.025–1.0 mM, L-leucine ϭ 0.3–10 mM, final concentrations) while keeping the other substrates constant. Studies of the structural models of bovine GDH were performed with the use of the RasMol (version 2.7.1.1, www.bernsteinplus-sons.com/software/rasmol), the Swiss-PDBviewer (version 3.7.b2, www.expasy.ch/spdbv), and the RIBBONS programs (24)
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