The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery

Peterson, P K; Smith, Thomas J.

doi:10.1016/s0969-2126(99)80101-4

Cited by 164 publications

(248 citation statements)

References 68 publications

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“…Previous studies have shown that GTP, a selective inhibitor of hGDH1, binds to both the closed and the open state of the enzyme, promoting a closed conformation (26). At the structural level, GTP is shown to occupy the space behind the NAD ϩ domain, when it rotates during catalysis to close the active site (26).…”

Section: Discussionmentioning

confidence: 98%

“…At the structural level, GTP is shown to occupy the space behind the NAD ϩ domain, when it rotates during catalysis to close the active site (26). This in turn prevents the NAD ϩ domain from moving The y axis shows the catalytic activity displayed by each recombinant enzyme prior to the addition of DES expressed as percentages of maximal activity.…”

Section: Discussionmentioning

confidence: 99%

“…back to open the catalytic cleft (26). To explore whether GTP binding alters the estrogen sensitivity of hGDH1, we obtained estrogen inhibitory curves in the presence of GTP.…”

Section: Discussionmentioning

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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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: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

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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“…The largest difference between the mammalian and bacterial GDH is the long antenna domain in mammalian GDH that is formed by the 48-amino acid insertion, beginning *To whom correspondence should be addressed. Tel: 82-33-248-2112;Fax: 82-33-241-1463 E-mail: sychoi@hallym.ac.kr at residue 395 (Peterson and Smith, 1999;Smith et al, 2001). The allosteric control of mammalian GDH activity by positive (ADP) and negative effectors (GTP) has been studied (Colman, 1991;Hudson and Daniel, 1993).…”

Section: Introductionmentioning

confidence: 99%

“…The mammalian GDH is strictly regulated by allosteric activators and inhibitors (McPherson and Wootton, 1983;Cho et al, 1995). GTP inhibits enzyme turnover over a wide range of conditions by increasing the affinity of the enzyme for the product, making product release rate-limiting under all conditions in the presence of ADP (Peterson and Smith, 1999;Smith et al, 2001). ADP is a potent activator that decreases product affinity (Bailey et al, 1982;Smith et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Molecular Gene Cloning, Expression, and Characterization of Bovine Brain Glutamate Dehydrogenase

Kim¹,

Eum²,

Jang³

et al. 2003

BMB Reports

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A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The K m and V max values for NAD + were 0.1 mM and 1.08 µmol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentrationdependent manner over the range of 10 -100 µM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

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Purification of Glutamate Dehydrogenase from Liver and Brain

et al. 2002

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Two alternative procedures are described for the purification of the major form of glutamate dehydrogenase (L-glutamate-NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3: GDH) from ox liver and brain. The first involves affinity chromatography on a column of the allosteric inhibitor GTP bound to Sepharose, whereas the other uses a bifunctional ligand (bis-NAD+) composed of two NAD+ molecules linked together by a spacer arm to precipitate the enzyme in the presence of the substrate analogue glutarate. In both procedures the affinity steps are preceded by ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose. Procedures for the synthesis of GTP-Sepharose and bis-NAD+ are described and the ancillary procedures, including the assay of GDH activity and the determination of protein concentration, are also presented.

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The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery

Cited by 164 publications

References 68 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

Molecular Gene Cloning, Expression, and Characterization of Bovine Brain Glutamate Dehydrogenase

Purification of Glutamate Dehydrogenase from Liver and Brain

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