Allosteric and substrate site effects of relevant amino acids on structural stability and flexibility of glutamate dehydrogenase-1 (GDH-1).

Abstract

Mammalian GDH-1, which converts glutamate to alpha-ketoglutarate and ammonia while reducing NAD(P), is constitutively expressed in virtually all cells of the body. The extensive literature on GDH-1 suggests that functionally relevant GDH-1 structures may be distributed over a conformational flexibility landscape: polyhexamer, hexamer, trimer, monomer, monomer (unfolded), monomer (misfolded), polymer (disassembled). We have used both intrinsic (tryptophan) and extrinsic (ANS) fluorescent probes to study the dependence of the reversible hexamer to trimer dissociation on the concentrations of the denaturing agents urea and guanidine hydrochloride (GdnHCl). Dissociation at each denaturant concentration increases with increase in concentration and/or temperature, and these dependencies are shown to be affected by amino acids which bind to allosteric and/or substrate binding sites. Dissociation of the hexamer is accompanied by a large increase in the fluorescence of ANS due to binding to increase in exposed hydrophobic microdomains on the protein. In contrast, tryptophan fluorescence decreases due to increased exposure of tryptopan in the protein to the aqueous environment. Measurement were made of the influence of several known substrates and allosteric modifiers (i.e. a selection physiologically relevant monocarboxylic amino acids) on the GDH-1 hexamer to trimer transition. The observed effect of adding each amino acid is consistent with independent measurements of its binding affinity. From the temperature dependence of the fluorescence intensity changes for ANS we obtained activation parameters for the hexamer-trimer transitions. Our data are consistent with the hexamer/trimer transition in GDH-1 having significant regulatory function in vivo, perhaps contribution to/complementing allosteric regulation of the enzyme activity.