Effect of the modification and denaturation of glutamate dehydrogenase on its polarographic behaviour

The peak occurring in the differential pulse polarogram of native bovine glutamate dehydrogenase (GDH) at about −1.5 V in neutral media is influenced by the modification of some amino acid residues of the enzyme. The reactions of GDH with acetic anhydride and diethylpyrocarbonate (which modify lysine and histidine residues, respectively, and decrease essentially the basicity of their nitrogens) result in a shift of the peak potential to more positive values. The reaction of GDH with ethanolamine in the presence of carbodiimide and N-hydroxysuccinimide (modification of carboxyl groups) causes a slight shift in the opposite direction. These effects, which are comparable to those exhibited by pH changes, suggest that the surface charge of the protein is important for the electrode reaction. On the other hand, the reactions of the sulfhydryl groups of the enzyme with p-chloromercuribenzoate and/or silver ions lead to a substantial decrease (or disappearance) of the peak. As the sensitivity of the peak height to these modifications surpasses that of enzyme activity, it is probable that some of the SH groups of the enzyme are responsible for the observed polarographic behaviour.The occurrence of the studied peak is connected with the preservation of the native structure of the protein since the denaturation of glutamate dehydrogenase in the presence of urea or in alkaline media brings about a parallel decrease in the peak height and in enzyme activity. The sensitivity of the described peak towards denaturation is its most conspicuous difference, in comparison with the properties of the Brdička current of glutamate dehydrogenase (which is detectable in the presence of Co(III) and can also be attributed to the evolution of hydrogen catalysed by sulfhydryl groups of this protein). The studied polarographic peak of glutamate dehydrogenase is not identical with its presodium current, which can be observed at comparable potentials in the presence of ammonia.