Characterization of a novel methanol dehydrogenase containing a Ba²⁺ ion at the active site

The quinoprotein methanol dehydrogenase (MDH) contains a Ca²⁺ ion at the active site. Ca²⁺-free enzyme (from a processing mutant) was used to 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 site. 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 µM) and the K_A for ammonia is 52 mM (compared with 2 mM). The different activity 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 binding and hence a decrease in activation energy. The kinetic model for Ba²⁺-MDH with respect to substrate and activator is consistent with previous 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 its low affinity for substrates, it is possible to prepare the oxidized form of Ba²⁺-MDH. No spectral intermediates could be detected during reduction by added substrate, and so it is not possible to distinguish between those mechanisms involving covalent substrate addition and those involving only hydride transfer.