SITE AND SIGNIFICANCE OF CHEMICALLY MODIFIABLE CYSTEINE RESIDUES IN GLUTAMATE-DEHYDROGENASE OF CLOSTRIDIUM-SYMBIOSUM AND THE USE OF PROTECTION STUDIES TO MEASURE COENZYME BINDING

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase ( GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experimen ts with various thiol-modifying reagents reveal that in native clostri dial GDH only one of these two cysteines is accessible for reaction. T his residue does not react with iodoacetate, iodoacetamide, N-ethylmal eimide or N-phenylmaleimide, but reaction with either p-chloromercurib enzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes comple te inactivation, preventable by NAD(+) or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates sh ow that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociatio n constant (0.69 mM) for the enzyme-NAD(+) complex. Similar data for N ADH indicated mildly cooperative binding with a Hill coefficient of 1. 32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources.