Identification and Characterization of Kinetically Competent Carbinolamine and α-Iminoglutarate Complexes in the Glutamate Dehydrogenase-Catalyzed Oxidation of l-Glutamate Using a Multiwavelength Transient State Approach

Abstract

A highly constrained and heavily overdetermined multiwavelength transient state kinetic approach has been used to study the oxidative deamination of L-glutamate catalyzed by beef liver glutamate dehydrogenase. Spectra generated using the known enzyme-reduced coenzyme-substrate spectrum served as models for deconvolution of kinetic scan data. Deconvolution of the multiwavelength time course array shows formation of three distinguishable intermediates in the reaction sequence, an ultrablue-shifted complex, an ultrared-shifted complex, and a blue-shifted complex. The ultrablue-shifted entity is identified as the enzyme-NADPH-alpha-iminoglutarate complex (ERI) and the ultrared as the enzyme-NADPH-alpha-carbinolamine complex (ERC). The blue-shifted complex is characterized as the E-NADPH-ketoglutarate species (ERK). The location of these species along the reaction coordinate has been determined and their kinetic competency in the reaction sequence has been established by fitting the concentration time courses of the components for both the alpha-deuterio- and the alpha-protio-L-glutamate reactions to the now highly constrained differential equations derived from a kinetic scheme involving the sequential formation of alpha-iminoglutarate, alpha-carbinolamine, and alpha-ketoglutarate-reduced coenzyme complexes, following the formation of two prehydride transfer complexes.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase

“…The NAD + -dependent bacterial/fungal enzymes are either homohexamers (13) or homo-tetramers (14,15). Despite numerous studies in the last 40 years to identify the intermediates formed during the reaction catalyzed by GDHs (16)(17)(18)(19)(20), structural basis of the reaction mechanism of this enzyme remains unresolved. A number of medium/low resolution crystal structures of GDHs have been determined as complexes with substrates (α-ketoglutarate or glutamate) and coenzymes (NADP + or NAD + ) (7,12,13,16,(21)(22)(23).…”

The Direct Measurement of Thermodynamic Parameters of Reactive Transient Intermediates of thel-Glutamate Dehydrogenase Reaction

“…Two recent experimental developments now provide the basis for the direct measurement of differences in thermodynamic parameters of spectroscopically identified reactive intermediate complexes whose kinetic competence has been verified. These developments are 1) the transient-state multiwavelength spectroscopic approach, which can produce resolved component reaction time courses of enzyme reactions (1,2); and 2) the Le Chatelier forced equilibrium approach, which produces significant concentrations of reactive transient intermediate complexes in solution under true thermodynamic equilibrium conditions (3,4). Here, we report the results of the first set of such thermodynamic measurements on reactive intermediates of the bovine liver L-glutamate dehydrogenase (blGDH) 1 reaction.…”

“…These developments are 1) the transient-state multiwavelength spectroscopic approach, which can produce resolved component reaction time courses of enzyme reactions (1,2); and 2) the Le Chatelier forced equilibrium approach, which produces significant concentrations of reactive transient intermediate complexes in solution under true thermodynamic equilibrium conditions (3,4). Here, we report the results of the first set of such thermodynamic measurements on reactive intermediates of the bovine liver L-glutamate dehydrogenase (blGDH) 1 reaction. The Experimental System-The stoichiometry of the glutamate dehydrogenase reaction is shown in Equation 1.…”

“…Here, we report the results of the first set of such thermodynamic measurements on reactive intermediates of the bovine liver L-glutamate dehydrogenase (blGDH) 1 reaction. The Experimental System-The stoichiometry of the glutamate dehydrogenase reaction is shown in Equation 1. The capital letter immediately below each reactant or product species is used to designate that entity in the various enzyme (E) complexes to be discussed.…”

“…7156); Fax: 816-861-1110; E-mail: hfisher@kumc.edu. 1 The abbreviations used are: blGDH, bovine liver GDH; GDH, glutamate dehydrogenase; E, enzyme; O, oxidized coenzyme; I, iminoglutarate; R, reduced coenzyme; G, L-glutamate; C, carbinolamine; EOG T , the sum of protonated and unprotonated EOG; K, ␣-ketoglutarate; EOG, enzymeoxidized coenzyme-L-glutamate complex; EOGЈ, isomerized EOG; HEOG, the protonated form of EOG; ER, the enzyme-NADPH complex; ERG, ER-L-glutamate; ERK, ER-␣-ketoglutarate; ERC, ER-␣-carbinolamine; ERI, ER-␣-iminoglutarate; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid, 5-enolpyruvoyl shikimate 3-phosphate. 2 Subsequent to publication of our paper (3) in which we introduced what we presumed to be a novel method and during the process of review of this paper, it came to our attention that the approach had in fact already been utilized successfully and elegantly in a paper from the laboratories of K. Johnson and K. Anderson in which they applied 13 C NMR to a forced equilibrium system to identify and characterize an enzyme-bound tetrahedral intermediate in the 5-enolpyruvoylshikimate 3-phosphate synthase reaction (4).…”