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 Vmax value is 2-fold higher. The affinities of Ba2+-MDH for substrate and activator are very much less than those of Ca2+-MDH; the Km for methanol is 3.5 mM (compared with 3 µM) and the KA 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.
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September 1996
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Research Article|
September 01 1996
Characterization of a novel methanol dehydrogenase containing a Ba2+ ion at the active site
Matthew G GOODWIN;
Matthew G GOODWIN
1Department of Biochemistry, Institute of Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Christopher ANTHONY
Christopher ANTHONY
*
1Department of Biochemistry, Institute of Biomolecular Sciences, University of Southampton, Southampton SO16 7PX, U.K.
*To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
March 07 1996
Revision Received:
May 09 1996
Accepted:
May 14 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 318 (2): 673–679.
Article history
Received:
March 07 1996
Revision Received:
May 09 1996
Accepted:
May 14 1996
Citation
Matthew G GOODWIN, Christopher ANTHONY; Characterization of a novel methanol dehydrogenase containing a Ba2+ ion at the active site. Biochem J 1 September 1996; 318 (2): 673–679. doi: https://doi.org/10.1042/bj3180673
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