Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations

Jie Fang, Betty Y L Hsu, Courtney M. MacMullen, Mortimer Poncz, Thomas Smith, Charles A. Stanley

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of L-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210 μM) and S448P (ED50, 3.1 μM) human GDH mutants compared with the wild-type human GDH (ED50, 42 nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit cooperativity of human GDH.

Original languageEnglish (US)
Pages (from-to)81-87
Number of pages7
JournalBiochemical Journal
Volume363
Issue number1
DOIs
StatePublished - Apr 1 2002
Externally publishedYes

Fingerprint

Glutamate Dehydrogenase
Purification
Guanosine Triphosphate
Mutation
Adenosine Diphosphate
Leucine
Chemical activation
Binding Sites
Nucleotides
Allosteric Site
Deamination
Mammals
X ray crystallography
X Ray Crystallography
Enzymes
Tail
Glutamic Acid
Adenosine Triphosphate
Insulin
Antennas

Keywords

  • Enzyme inhibitor
  • Gene expression regulation
  • Hyperammonaemia
  • Hyperinsulinism
  • Protein structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. / Fang, Jie; Hsu, Betty Y L; MacMullen, Courtney M.; Poncz, Mortimer; Smith, Thomas; Stanley, Charles A.

In: Biochemical Journal, Vol. 363, No. 1, 01.04.2002, p. 81-87.

Research output: Contribution to journalArticle

Fang, Jie ; Hsu, Betty Y L ; MacMullen, Courtney M. ; Poncz, Mortimer ; Smith, Thomas ; Stanley, Charles A. / Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. In: Biochemical Journal. 2002 ; Vol. 363, No. 1. pp. 81-87.
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AU - Stanley, Charles A.

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AB - Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of L-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210 μM) and S448P (ED50, 3.1 μM) human GDH mutants compared with the wild-type human GDH (ED50, 42 nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit cooperativity of human GDH.

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