title: Positive cooperativity with Hill coefficients of up to 6 in the glutamate concentration dependence of steady-state reaction rates measured with clostridial glutamate dehydrogenase and the mutant A163G at high pH.
abstract: Glutamate dehydrogenases from many sources display nonclassical kinetic behavior suggestive of allosteric interaction among the six subunits of the hexamer. A three-dimensional structure now potentially offers a framework for explaining the basis of such behavior in clostridial glutamate dehydrogenase, and this paper offers evidence of extreme, all-or-none cooperativity in the binding of glutamate by this enzyme. A site-directed mutant of clostridial glutamate dehydrogenase in which Ala163 in the glutamate binding site is replaced by glycine displays a markedly sigmoid dependence of reaction rate on glutamate concentration (S0.5 = 200 mM), with a Hill coefficient of 3.4 when assayed at pH 10.5 with 1 mM NAD+. Under the same conditions the wild-type enzyme gave no measurable rate with glutamate concentrations in the range normally used for kinetics (0-100 mM) but gave a steep rise in reaction rate from 600 to 1200 mM glutamate. At pH 9.0, where the wild-type enzyme has previously been shown to be "inactive" in a standard assay, a study extending to much higher glutamate concentrations again revealed a sigmoid dependence, with a Hill coefficient of 5.4 and an S0.5 at 150 mM glutamate. With the mutant A163G the apparent cooperativity was less, with a Hill coefficient of 2.3, and the affinity for glutamate was higher, with S0.5 of 7 mM. Both proteins gave normal hyperbolic dependence on glutamate concentration at pH 7 and pH 8. At pH 9 and with saturating glutamate, both enzymes showed a hyperbolic dependence of the rate on NAD+ concentration. The NAD+ concentration, however, affected the observed degree of cooperativity with varied glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University College Dublin, Belfield, Ireland.
authors.forename: X G
authors.initials: XG
authors.lastname: Wang
authors.forename: P C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D000494:Allosteric Regulation; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D013379:Substrate Specificity; D013386:Succinates / Q000737:chemistry; D019802:Succinic Acid
chemical_list: D017931:DNA Primers; D013386:Succinates; D009243:NAD; D018698:Glutamic Acid; D019802:Succinic Acid; D005969:Glutamate Dehydrogenase