Reversible reduction of an .alpha.-imino acid to an .alpha.-amino acid catalyzed by glutamate dehydrogenase: effect of ionizable functional groups

Abstract

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)

Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase

“…The reaction mechanism of GDH has been extensively studied using a variety of methods [36–38]. Previous chemical modification studies have also suggested that the essential reactive Lys residues may be involved in the GDH reaction [12,39].…”

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery

“…The chemical mechanism of GDH has been extensively studied using a variety of methods [7,[10][11][12]. For boGDH, it has been proposed that closing the active-site cleft provides a hydrophobic environment suitable for hydride transfer [12].…”

“…On the basis of sequence similarities to csGDH [7], Lys113 and Lys90 are proposed to be involved in salt bridges to the αand γ-carboxyl groups of the substrate, respectively. In the proposed model for catalysis [11,12], Lys126, which has an abnormally low pK a , spontaneously loses an H + to solvent allowing the cleft to close and triggering three subsequent events: a single water molecule becomes hydrogen bonded to the NH2 group of Lys126; the bulk solvent is expelled from the cleft; and the α-hydrogen atom of the substrate is brought into the correct orientation and position to permit hydride transfer to the nicotinamide ring of NAD(P) + . The major difference between boGDH and csGDH is that, whereas the bovine form releases the Lys126 proton to solvent prior to hydride transfer, this apparently occurs after hydride transfer in csGDH [12][13][14].…”

Comparison of the energetics of the uncatalyzed and glutamate dehydrogenase catalyzed .alpha.-imino acid-.alpha.-amino acid interconversion

“…The reduction of PCA is also catalyzed by glutamate de-hydrogenase2 (where only the L isomer of the amino acid participates in the reaction), and the rates are now measurable in the forward and reverse directions (Fisher et al, 1982;Srinivasan & Fisher, 1985a). The enzyme-catalyzed reaction, representing the reversible reduction of PCA by the enzyme-NADPH complex, is shown in eq 2.…”