Kinetic studies of the active sites functioning in the quinohemoprotein fructose dehydrogenase.

Steady-state kinetic analysis was performed on the reaction between D-fructose and ferricyanide with the quinohemoprotein fructose dehydrogenase from Gluconobacter species. The D-fructose oxidation dependence on the ferricyanide concentration resulted in a series of parallel reciprocal plots, and the reaction was assumed to proceed by a ping-pong type of mechanism. A reciprocal plot of the reduction of ferricyanide at saturating concentration of D-fructose gave a break which was considered to appear as a result of the two active centers, namely PQQ and heme c functioning. A scheme of action is proposed and the bimolecular rate constant of the D-fructose oxidation, the kcat for PQQ and the electron transfer rate between the PQQH2 and heme c are calculated and account for 2.2 +/- 0.4 x 10(4) M-1 s-1, (93 +/- 14) and (162 +/- 7) s-1, respectively.