Ligand-induced alterations in the reactivity of sulfhydryl groups and the structure of bovine liver glutamate dehydrogenase

The fluorogenic probe 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) was employed as an environmentally sensitive reporter label for free sulfhydryl groups of bovine liver glutamate dehydrogenase. A maximum of six -SH groups per subunit was titrated in Tris and borate buffers (pH 7.8) in 2 h but there was no reaction in the presence of phosphate buffer. The rate and extent of -SH reactivity was changed significantly by one of the substrates and some allosteric effectors. Adenosine nucleotides, NADH, and α-ketoglutarate promoted conformational alterations in glutamate dehydrogenase such that -SH groups were rendered virtually unreactive in [enzyme-ADP], [enzyme-NADH], and [enzyme-α-ketoglutarate]binary complexes. GTP, a negative allosteric modulator, showed no effect on -SH exposure. Measurements of protein circular dichroism spectra and catalytic activity in conjunction with -SH reactivity demonstrated a direct relationship between structural stability, biological activity, and ligand-induced conformational changes. The ligands that strongly protected the enzyme from reaction with NBD-C1 concomitantly maintained its structural and functional integrity.