Identification and characterization of kinetically competent carbinolamine and alpha-iminoglutarate complexes in the glutamate dehydrogenase-catalyzed oxidation of L-glutamate using a multiwavelength transient state approach.
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.
Laboratory of Molecular Biochemistry, VA Medical Center, Kansas City, Missouri 64128, USA.
S J
SJ
Maniscalco
S K
SK
Saha
H F
HF
Fisher
D000465:Algorithms; D000818:Animals; D002384:Catalysis; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D005977:Glutarates / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry*; D007098:Imino Acids / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
D005977:Glutarates; D007098:Imino Acids; D046911:Macromolecular Substances; C054902:iminoglutarate; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D006836:Hydro-Lyases; C076392:pterin-4a-carbinolamine dehydratase