The Effects of Leucine on Regulation of Glutamate Dehydrogenase by Purine Nucleotides

Abstract

The activity of glutamate dehydrogenase (GDH) is regulated by a complex interplay of homotropic ( cofactors ) and heterotropic (including purine nucleotides and leucine) allosteric effects. The aim of this study was to examine the allosteric regulation of GDH in an ”in vitro” context to more closely parallel cellular environments. Most previous studies of this complex enzyme have involved investigating the effects of single regulatory molecules on activity but the aim of the current project is to assess whether particular regulatory ligands modulate the effects of others. We examined the effects of pH, GTP, ADP and cofactor choice (NADP vs NAD) on leucine regulation of GDH activity. Kinetics studies showed that the activation by leucine on GDH had an optimum pH of 8, that cofactor NAD had greater activity than cofactor NADP, that GTP inhibited in the presence of both cofactors but was reversed differentially by leucine, and activity increased as cofactor saturation increased. To investigate the mechanism of these allosteric effects, CD in conjunction with thermal melts was used to analyze the stability of GDH in the presence and absence of leucine. The results analyzed using a 4 parameter Hill equation indicate that leucine significantly raises the Tm and the cooperativity of folding increases suggesting tighter coupling between different regions of the molecule.