Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase

Abstract

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5 H -phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent V max of the Lys130 mutant enzymes, apparent K m values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata

“…It also indicates that the different sensitivities to alkalized extracts in the presence of high concentration of ADP may due to differences in the regulatory properties between hGDH isozymes by ADP. These observations are consistent with the previous reports that there are at least two different GDH activities differing in their relative thermal stability and allosteric regulation characteristics (Mavrothalassitis et al, 1988;Shashidharan et al, 1994;Cho et al, 2001;Yang et al, 2004). Since physiological ADP levels can vary from 0.05 to 1.0 mM depending on the rate of oxidative phosphorylation, our results suggest a possibility that hGDH1 and hGDH2 are differently regulated in vivo by the actions of alkalized extracts depending on the physiological concentrations of ADP.…”

Identification of the GTP Binding Site of Human Glutamate Dehydrogenase by Cassette Mutagenesis and Photoaffinity Labeling

“…Human GDH gene (pHGDH) has been chemically synthesized and expressed in E. coli as a soluble protein in our laboratory as described elsewhere (23). All other chemicals and solvents were reagent grade or better.…”

“…Mutants-A series of single amino acid substitutions of Tyr-266 or Lys-450 was constructed separately by cassette mutagenesis of a synthetic human GDH gene, pHGDH (23). For, the Tyr-266 site, plasmid DNA (5 g) was digested with StuI and NsiI to remove a 42-bp fragment that encodes amino acids 260 -273; this fragment was replaced with five 42-bp synthetic DNA duplexes containing a substitution on both DNA strands at positions encoding Tyr-266 to make Y266G, Y266S, Y266E, Y266M, and Y266R.…”

“…Cell pellets were suspended in 100 ml of 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM dithiothreitol and lysed with a sonicator. The wild type GDH and the mutant GDHs were purified by the method developed in our laboratory (23,27). Because the wild type GDH and the mutant GDHs were readily solubilized, no detergents were required throughout the entire purification steps.…”

“…Very recently, a 1557-base pair gene that encodes human GDH has been synthesized and expressed in Escherichia coli in our laboratory (23). For the present study, the mutant human GDHs, containing Glu, Gly, Met, Ser, or Arg at the Tyr-266 site and Glu, Gly, Met, Ser, or Tyr at the Lys-450 site have been expressed in E. coli as a soluble protein, purified, and characterized.…”