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

Biochemistry
X G Wang, P C Engel

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 cooperativ...Continue Reading

Citations

Apr 16, 1998·Biochimica Et Biophysica Acta·L A BassoA R Walmsley
Dec 18, 2001·Biophysical Chemistry·M F TestorfI Lundström
Mar 23, 2011·Neurochemistry International·Paul C Engel
May 9, 2009·Proteins·Michael A Sharkey, Paul C Engel
Mar 25, 2011·Biochemical Society Transactions·Paul C Engel
Jun 16, 2016·Acta Crystallographica. Section F, Structural Biology Communications·Tânia OliveiraAmir R Khan
Jul 29, 2017·American Journal of Psychoanalysis·Marcio Leitão Bandeira
Nov 5, 2002·The Journal of Biological Chemistry·Suren AghajanianPaul C Engel
Mar 16, 2018·The Journal of Biological Chemistry·Prem PrakashPrasenjit Bhaumik

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