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

The Biochemical Journal
Jie FangCharles A Stanley

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 (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cel...Continue Reading

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Citations

Oct 12, 2010·Reviews in Endocrine & Metabolic Disorders·Andrew A Palladino, Charles A Stanley
Jun 11, 2002·Journal of Molecular Biology·Thomas J SmithCharles A Stanley
Oct 10, 2009·PLoS Computational Biology·Omar N A DemerdashJulie C Mitchell
Jul 26, 2008·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Riko D KlootwijkMarjan Huizing
Jul 25, 2009·The American Journal of Clinical Nutrition·Charles A Stanley
Dec 14, 2011·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Saleh UmairHeather V Simpson
Aug 2, 2011·Journal of Proteomics·Jia GuoLaszlo Prokai
May 7, 2009·Journal of Neurochemistry·Konstantinos KanavourasAndreas Plaitakis
Jul 29, 2010·Protein Science : a Publication of the Protein Society·Sarah A WackerEllis Bell
Jan 3, 2013·Glia·Daniel KomlosBonnie L Firestein
Mar 7, 2013·Journal of Neuroscience Research·Andreas PlaitakisCleanthe Spanaki
Oct 4, 2005·Archives de pédiatrie : organe officiel de la Sociéte française de pédiatrie·I GiurgeaP de Lonlay
Sep 11, 2010·Microbiology·Takeo TomitaMakoto Nishiyama
Dec 9, 2003·Journal of Biochemistry and Molecular Biology·Dae Won KimSoo Young Choi
Jun 16, 2015·British Journal of Clinical Pharmacology·A D Bastiaan VliegenthartJames W Dear
Feb 12, 2008·Biochemical and Biophysical Research Communications·Myung-Min ChoiSung-Woo Cho
Nov 15, 2011·Metabolism: Clinical and Experimental·Priscilla Muniz Ribeiro da SilvaEverardo Magalhães Carneiro
Dec 6, 2012·Acta Biochimica Et Biophysica Sinica·Tingting Zhang, Changhong Li
Jan 6, 2009·International Journal of Biological Macromolecules·Marjan SabbaghianMohsen Nemat-Gorgani
May 23, 2013·Molecules and Cells·Sang Gyun KimJohn Blenis
Aug 24, 2002·The Journal of Biological Chemistry·Hye-Young YoonSung-Woo Cho
Apr 1, 2006·The Journal of Biological Chemistry·Changhong LiCharles A Stanley
Sep 23, 2010·Journal of Pediatric Endocrinology & Metabolism : JPEM·Chengming DiaoHeng Wang

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