title: The direct measurement of thermodynamic parameters of reactive transient intermediates of the L-glutamate dehydrogenase reaction.
abstract: In a previous report (Fisher, H. F., Maniscalco, S. J., and Tally, J. (2002) Biochem. Biophys. Res. Commun. 287, 343-347) we demonstrated the capability of the "Le Chatelier forcing method" of producing stable solutions containing substantial amounts of transitory enzyme intermediate complexes that can otherwise be observed only fleetingly in the millisecond time range. The method requires nothing more than running an enzyme reaction using forcing concentrations of reactants against an equally forcing concentration of products until equilibrium is attained. Here we have applied this approach to the measurement of the thermodynamics of several such reactive (and normally transient) intermediate complexes of the bovine liver l-glutamate dehydrogenase-catalyzed reaction. At pH 9.5 and 20 degrees C, we observe both the enzyme-NADPH-alpha-iminoglutarate and enzyme-NADPH-alpha-carbinolamine complexes at concentrations whose sum accounts for 70% of the total enzyme. The pH dependence of these two complexes under equilibrium conditions provides thermodynamic parameters for both the protonated and the unprotonated forms of each of these two entities as well as those of the enzyme-NADP-l-glutamate complex. The equilibrium concentrations of each of these reactive complexes are compared with their corresponding transient steady-state values.
authors.affiliation: Laboratory of Molecular Biochemistry, Veteran Affairs Medical Center and the Department of Biochemistry, University of Kansas Medical Center, 4801 Linwood Boulevard, Kansas City, MO 64128, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Maniscalco
authors.forename: Jon F
authors.initials: JF
authors.lastname: Tally
authors.forename: S Welsh
authors.initials: SW
authors.lastname: Harris
authors.forename: Harvey F
authors.initials: HF
authors.lastname: Fisher
mesh_terms: D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013816:Thermodynamics
chemical_list: D005969:Glutamate Dehydrogenase