Polarographic study of horse liver alcohol dehydrogenase lacking zinc ions at the active sites
Kovar, J.; Klukanova, H.; Studnickova, M.; Zeppezauer, M.; Maret, W.
Bioelectrochemistry and Bioenergetics 13(4-6): 407-416
1985
DOI: 10.1016/0302-4598(84)87042-7
Accession: 006138010
In the differential pulse polarogram of this enzyme only one peak (at the potential of -1.1 V) was detected. The behaviour of this peak under changing polarographic conditions was similar to that of one of the two peaks of native alcohol dehydrogenase (observed at the same potential); this confirmed the role of the structural zinc of the studied enyme and of native alcohol dehydrogenase in the occurrence of the signal. The denaturation of the enzyme depleted of catalytic zinc brought about an increase in the current at -1.0 V (reduction of liberated Zn2+ ions) whereas the peak at -1.1 V disappeared. The titration of the denatured enzyme with standard additions of ZnSO4 confirmed the stoichiometry of one zinc ion per enzyme subunit. The analysis of differential pulse polarograms of the zinc-depleted enzyme in the presence of increasing ZnSO4 concentrations revealed that this enzyme is able to bind Zn2+ ions (at variance with the native enzyme). The differential pulse polarograms of the native enzyme and of that without the catalytic zinc in the Brdicka solution were similar. The difference in the currents at -1.4 V suggests that the additional sulfhydryl groups located in a hydrophobic region of the zinc-depleted enzyme might be active in the Brdicka reaction.