Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca(2+)-free enzyme with Ca2+, Sr2+ or Ba2+.
The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per alpha 2 beta 2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.
Goodwin
M G
MG
Department of Biochemistry, School of Biological Sciences, University of Southampton, U.K.
Avezoux
A
A
Dales
S L
SL
Anthony
C
C
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001051:Apoenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001464:Barium / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013053:Spectrophotometry; D013324:Strontium / Q000378:metabolism*; D013816:Thermodynamics
D001051:Apoenzymes; D001464:Barium; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D002118:Calcium; D013324:Strontium