Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca(2+)-free enzyme with Ca2+, Sr2+ or Ba2+

Biochem J. 1996 Nov 1;319 ( Pt 3)(Pt 3):839-42. doi: 10.1042/bj3190839.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / biosynthesis
  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / metabolism*
  • Apoenzymes / biosynthesis
  • Apoenzymes / chemistry*
  • Apoenzymes / metabolism*
  • Barium / metabolism*
  • Calcium / metabolism*
  • Enzyme Activation
  • Genes, Bacterial
  • Gram-Negative Aerobic Bacteria / enzymology*
  • Gram-Negative Aerobic Bacteria / genetics
  • Hydrogen-Ion Concentration
  • Kinetics
  • Spectrophotometry
  • Strontium / metabolism*
  • Thermodynamics

Substances

  • Apoenzymes
  • Barium
  • Alcohol Oxidoreductases
  • alcohol dehydrogenase (acceptor)
  • Calcium
  • Strontium