Reversible reduction of an alpha-imino acid to an alpha-amino acid catalyzed by glutamate dehydrogenase: effect of ionizable functional groups.
The glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (PCA; an alpha-imino acid) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) to give L-proline and NADP+ is employed as a model for the redox step of the corresponding enzyme-catalyzed reductive amination of alpha-ketoglutarate. We demonstrate the reversibility of the model reaction and measure its equilibrium constant. The pH profiles for the model reactions show that the active substrates are the N-protonated imino acid in one direction and the proline anion with a neutral amino group in the other. The V/K value for the imino acid reduction is enhanced by a group Z of pK = 8.6 in the enzyme-NADPH complex, while that for the proline reaction is unaffected by any such group in the enzyme-NADP+ complex. The following conclusions emerge from a comparison of the pH dependence of the rates for the model reactions with that for the oxidative deamination of L-glutamate [Rife, J. E., & Cleland, W. W. (1980) Biochemistry 19, 2328]. The N-protonated form of alpha-iminoglutarate and the conjugate base of glutamate are the active substrates. The redox step is not sensitive to the protonation state of the groups that catalyze the hydrolysis of bound alpha-iminoglutarate. The group Z, which facilitates the PCA reaction, plays no role in the binding of alpha-ketoglutarate. We propose a chemical mechanism for the glutamate reaction where an unprotonated enzyme group of pK = 5.2 in enzyme-NADPH catalyzes the conversion of the alpha-iminoglutarate to the carbinolamine.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011392:Proline / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism*
D011758:Pyrroles; C015485:delta-1-pyrroline-5-carboxylate; D009249:NADP; D011392:Proline; D005969:Glutamate Dehydrogenase