title: Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.
abstract: Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
authors.forename: S E
authors.initials: SE
authors.lastname: Syed
authors.forename: D P
authors.initials: DP
authors.lastname: Hornby
authors.forename: P E
authors.initials: PE
authors.lastname: Brown
authors.forename: J E
authors.initials: JE
authors.lastname: Fitton
authors.forename: P C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism*; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D013438:Sulfhydryl Compounds
chemical_list: D000596:Amino Acids; D003067:Coenzymes; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D009243:NAD; D005969:Glutamate Dehydrogenase; D003545:Cysteine