Kinetics and mechanism of the dye-coupleed cytoplasmic polyol dehydrogenase from Gluconobacter melanogenus

Abstract

A few form of the cytoplasmic polyol dehydrogenase having a distinct cofactor and substrate specificity was isolated from the cell free extracts of the sorbitol grown cells of $\mbox{\underline{G.}}$ $\mbox{\underline{melanogenus}}$ and purified partially by a CM-cellulose and a Sephadex G-100 column. The enzyme was found to be a dye-coupled oxidoreductase unlike all the other known cytoplasmic polyol dehydrogenases, and its catalytic activity was demonstrated only in the presence of an artificial electron acceptor like DPIP. The enzyme did not show any requirement of nicotinamide nucleotide coenzymes, $NAD^{+}$ or $NADP^{+}$, or molecular oxygen for the enzymatic polyol oxidation. The enzyme showed a very restricted substrate specificity toward the polyols having D-lyxo configuration such as D-mannitol and D-sorbitol. Stoichiometric composition of the polyol and DPIP for this enzyme catalyzed reaction showed a linear quantitative relationship between the ketose formation and the DPIP reduction. The steady-state kinetic studies on the dye-coupled polyol-dehydrogenase was carried out by the initial velocity measurements in the polyol oxidation and by the product inhibition analysis. The product inhibition patterns obtained by D-fructose (one no-inhibition, one noncompetitive, and two competitive) as well as the initial rate experiments showing the typical parallel pattern in the double reciprocal plot may provide the confirmatory evidences that the polyol oxidation by the enzyme proceeds by a ping-pong bi-bi mechanism. Based on the kinetic parameters obtained, it was also suggested that a rate-limiting step of the enzymatic polyol oxidation is associated with release of the ketose from the enzyme-polyol complex. The involvement of some essential side chain groups for the enzyme catalysis was suggested based on the results of the experiment on the pH dependency of the kinetic parameters, Kma and Vmax, of the enzyme reaction and the chemical modifications inclu...