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. Ca(2-)-free enzyme (from a processing mutant) was used to obtain enzyme containing Sr2+ or Ba2+, the Ba(2+)-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 Ba(2+)-MDH is less than half that of the reaction catalysed by Ca(2+)-MDH (a difference of 21.4 kJ/mol), and the Vmax value is 2-fold higher. The affinities of Ba(2+)-MDH for substrate and activator are very much less than those of Ca(2+)-MDH; the Km for methanol is 3.5 mM (compared with 3 microM) and the KA for ammonia is 52 mM (compared with 2 mM). The different activity of Ba(2+)-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 Ba(2+)-MDH with respect to substrate and activator is consistent with previous models for Ca(2+)-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 Ba(2+)-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.
Goodwin
M G
MG
Department of Biochemistry, University of Southampton, U.K.
Anthony
C
C
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D001464:Barium / Q000032:analysis* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D019605:Euryarchaeota / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D045542:PQQ Cofactor; D015363:Quinolones / Q000378:metabolism; D013053:Spectrophotometry; D013324:Strontium / Q000378:metabolism
D015363:Quinolones; D000643:Ammonium Chloride; D001464:Barium; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D002118:Calcium; D013324:Strontium