QUINOPROTEIN ETHANOL DEHYDROGENASE - PREPARATION OF THE APO-FORM AND RECONSTITUTION WITH PYRROLOQUINOLINE QUINONE AND CA2+ OR SR2+ IONS

Homogeneous quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 contains one Ca2+ ion per subunit of native enzyme. Treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid at 30-degrees-C led to an catalytically inactive apo-form. Upon incubation of the apo-form with Ca2+ and pyrroloquinoline quinone a fully active holo-enzyme was reconstituted. The absorption spectrum of this reconstituted enzyme is identical to that of the native enzyme. Incubation of apo-enzyme with Sr2+ and PQQ led to the formation of an active Sr2+-form. The Sr2+ and the (Ca2+-forms of the enzyme differ in their absorption spectra. The Sr2+-form was inactivated by trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid twice as fast as the Ca2+-form. Ca2+ is necessary for pyrroloquinoline quinone to bind to the apo-form of quinoprotein ethanol dehydrogenase.