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

Zaganas, Ioannis; Spanaki, Cleanthe; Karpusas, Michael; Plaitakis, Andreas

doi:10.1074/jbc.m208596200

Cited by 46 publications

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“…Structural analyses of the R443S mutant have suggested that introduction of Ser 443 disrupts side chain bonds in the antenna between Arg 443 of one subunit and Ser 409 of the adjacent subunit, leading to a closed conformation (15). This possibility is supported by additional studies 3 showing that substituting Arg for Ser 409 , which also disrupts the above side chain bonds, diminishes basal activity.…”

Section: Discussionsupporting

confidence: 63%

“…Initially, we studied the S409(R/D) hGDH1 mutants, which, similar to the R443S hGDH1 mutant, show low basal activity. 3 Introduction of Arg at residue 409 is thought to disrupt side chain bonds in the antenna region between Arg 443 of one subunit and Ser 409 of the adjacent subunit (15). Functional analyses of the expressed enzyme, purified to homogeneity, revealed that the S409R-hGDH1 mutant was indeed much more sensitive to estrogens than the wild-type hGDH1 (Fig.…”

Section: Study Of Hgdh1 and Hgdh2 Mutants Reveals That Sensitivity Tomentioning

confidence: 99%

“…Although hGDH1 is potently inhibited by GTP (IC 50 ϭ 0.1-0.3 M), hGDH2 has dissociated its function from GTP control (IC 50 ϭ ϳ100 M) because of evolutionary replacement of Gly 456 by Ala (14). Instead, regulation of hGDH2 is achieved via a novel molecular mechanism that mainly resulted from evolutionary substitution of Ser for Arg 443 (15). In the absence of allosteric effectors, hGDH2 assumes a closed state associated with little catalytic activity while remaining remarkably responsive to activation by ADP and/or L-leucine (16).…”

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Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Borompokas

Papachatzaki

Kanavouras

et al. 2010

Journal of Biological Chemistry

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Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC 50 ‫؍‬ 0.1-0.3 M) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC 50 ‫؍‬ 0.08 ؎ 0.01 M) and with ϳ18-fold lower affinity hGDH1 (IC 50 ‫؍‬ 1.67 ؎ 0.06 M; p < 0.001). Similarly, 17␤-estradiol showed a ϳ18-fold higher affinity for hGDH2 (IC 50 ‫؍‬ 1.53 ؎ 0.24 M) than for hGDH1 (IC 50 ‫؍‬ 26.94 ؎ 1.07 M; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R ‫؍‬ 0.99; p ‫؍‬ 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.

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Section: Discussionsupporting

confidence: 63%

Section: Study Of Hgdh1 and Hgdh2 Mutants Reveals That Sensitivity Tomentioning

confidence: 99%

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Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Borompokas

Papachatzaki

Kanavouras

et al. 2010

Journal of Biological Chemistry

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“…Conversely, to achieve full-range regulation by these activators, hGDH2 needs to set its basal activity at low levels (about 4-8% of full capacity), a property largely conferred by the evolutionary Arg443Ser change. 29 Hence, the detected Ser445Ala change has altered a property that is crucial for the tight regulation of hGDH2. 13 Substitution of Ala for Ser445 is predicted by secondary structure prediction programs 30 to stabilize the small á-helix of the antenna.…”

Section: Discussionmentioning

confidence: 99%

Gain-of-function variant in GLUD2 glutamate dehydrogenase modifies Parkinson's disease onset

et al. 2009

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Parkinson's disease (PD), a common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and their terminations in the basal ganglia, is thought to be related to genetic and environmental factors. Although the pathophysiology of PD neurodegeneration remains unclear, protein misfolding, mitochondrial abnormalities, glutamate dysfunction and/or oxidative stress have been implicated. In this study, we report that a rare T1492G variant in GLUD2, an X-linked gene encoding a glutamate dehydrogenase (a mitochondrial enzyme central to glutamate metabolism) that is expressed in brain (hGDH2), interacted significantly with age at PD onset in Caucasian populations. Individuals hemizygous for this GLUD2 coding change that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 developed PD 6-13 years earlier than did subjects with other genotypes in two independent Greek PD groups and one North American PD cohort. However, this effect was not present in female PD patients who were heterozygous for the DNA change. The variant enzyme, obtained by substitution of Ala for Ser445, showed an enhanced basal activity that was resistant to GTP inhibition but markedly sensitive to modification by estrogens. Thus, a gain-of-function rare polymorphism in hGDH2 hastens the onset of PD in hemizygous subjects, probably by damaging nigral cells through enhanced glutamate oxidative dehydrogenation. The lack of effect in female heterozygous PD patients could be related to a modification of the overactive variant enzyme by estrogens. INTRODUCTIONParkinson's disease (PD) affects about 1.8% of people over the age of 65 years. 1 It is clinically characterized by tremor, rigidity and bradykinesia, which often occur along with disturbances of posture and gait. 2 About 80% of PD cases are sporadic, with males being more frequently affected than females (ratio¼1.5:1).The etiology of PD remains largely unknown, although interplay of environmental toxins with genetic susceptibility may be operational. The genetic hypothesis is supported by community-based studies showing that a substantial number of PD patients have similarly affected relatives. 3,4 This was also revealed by a PD study conducted by us on Crete, 5 an island of about 0.6 million people sharing the same genetic and cultural background and a common environment. Moreover, this study suggested an oligogenic model for PD, according to which disease-predisposing alleles from two or more genes need to be present in the same individual for the disorder to be expressed.With regard to the pathophysiology of PD, several hypotheses have been proposed in an attempt to explain the degeneration of dopaminergic neurons in this disorder. One of these suggests that mitochondrial dysfunction is involved, 6 a contention supported by the recent discovery of monogenetic forms of PD resulting from mutations of

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“…Reciprocally interchanging amino acids that are different between hGDH isozymes within the regulatory domain may reveal the residues important for preference in hGDH isozymes. Recent studies of structure-function relationships using site-directed mutagenesis of hGDH1 at single sites differing from hGDH2 showed that the R443S and the G456A change reproduced some but not all of the properties of hGDH2 (3)(4)(5)17). In addition, several other residues differing between hGDH isozymes also have been examined by many investigators (4,17).…”

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Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

Choi

Kim

Yang

et al. 2007

Journal of Biological Chemistry

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Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2 390 -448 )hGDH1 (amino acid segment 390 -448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1 390 -448 )hGDH2 (amino acid segment 390 -448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and L-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

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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

Cited by 46 publications

References 36 publications

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Gain-of-function variant in GLUD2 glutamate dehydrogenase modifies Parkinson's disease onset

Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

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