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.
Glutamate dehydrogenase from beef liver (bl GDH) and the corresponding enzyme from Clostridium symbiosum (cs GDH) each catalyze the same sequence of chemical events in the oxidative deamination of L-glutamate. This catalysis involves interactions between at least six conserved functional groups, each of which appears to occupy the same geometric position with respect to the substrate molecule in both enzyme--coenzyme--L-glutamate reactive ternary complexes. In both cases steady-state V/K pH profiles indicate the requirement for the transfer to the solvent of a single proton from the same abnormal lysine for L-glutamate to bind and react; the pK of that lysine is the same for both enzymes. Here we report studies of the proton traffic between enzyme and solvent using direct pH-stat back-titration and indicator dye measurements on dead-end inhibitor ternary complexes, simultaneous transient-state time courses of proton and product, and transient-state kinetic isotope studies on both enzymes. We find that in the cs GDH catalyzed reaction the single proton is released only after the hydride transfer step whereas in the bl GDH reaction this proton release occurs prior to the hydride transfer step, despite the fact that the substrate molecule undergoes the same sequence of chemical events in both reactions. Interpreting these results in the context of the X-ray crystallographic structures of cs GDH and its NAD binary complex and of thermodynamic studies of bl GDH and its complexes, we conclude that the difference in the relative times of proton release in the two enzyme-catalyzed reactions must be ascribed to a difference in the sequence of active site cleft-opening and -closing events in the two identical reaction sequences. We suggest a possible biological significance to this unusual method of modulating a common reaction to suit differing metabolic roles.
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