CHARACTERIZATION OF A NOVEL METHANES DEHYDROGENASE CONTAINING A BA2-SITE( ION AT THE ACTIVE)

The quinoprotein methanol dehydrogenase (MDH) contains a Ca2+ ion at t he active site. Ca2+-free enzyme (from a processing mutant) was used t o obtain enzyme containing Sr2+ or Ba2+, the Ba2+-MDH being the first enzyme to be described in which a Ba2+ ion functions at the active sit e. The activation energy for oxidation of methanol by Ba2+-MDH is less than half that of the reaction catalysed by Ca2+-MDH (a difference of 21.4 kJ/mol), and the V-max value is 2-fold higher. The affinities of Ba2+-MDH for substrate and activator are very much less than those of Ca2+-MDH; the K-m for methanol is 3.5 mM (compared with 3 mu M) and t he K-A for ammonia is 52 mM (compared with 2 mM). The different activi ty of Ba2+-MDH is probably due to a change in the conformation of the active site, leading to a decrease in the free energy of substrate bin ding and hence a decrease in activation energy. The kinetic model for Ba2+-MDH with respect to substrate and activator is consistent with pr evious models for Ca2+-MDH. The pronounced deuterium isotope effect (6 .0-7.6) is influenced by ammonia, and is consistent with activation of the pyrroloquinoline quinone reduction step by ammonia. Because of it s low affinity for substrates, it is possible to prepare the oxidized form of Ba2+-MDH. No spectral intermediates could be detected during r eduction by added substrate, and so it is not possible to distinguish between those mechanisms involving covalent substrate addition and tho se involving only hydride transfer.