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Negative cooperativity in the steady-state kinetics of sugar oxidation by soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus.

Steady-state-kinetics investigations were carried out for the oxidation of aldose sugars by soluble quinoprotein glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus using N-methylphenazonium methyl sulfate (PMS) as artificial electron acceptor. As is not uncommon for a dye-linked dehydrogenase, the enzyme showed ping-pong behaviour and double-substrate inhibition. However, under conditions that avoided its masking by sugar-substrate inhibition as much as possible, negative kinetic cooperativity with respect to sugar substrate oxidation by this enzyme was demonstrated. Arguments are presented that exclude trivial factors as a cause for the phenomenon observed. Experimental data could be fitted with an equation accounting for biphasic cooperativity containing two sets of apparent kinetic parameters, V1 and K1, and V1 and K2, representing the enzyme's Michaelis-Menten behaviour at low and high substrate concentrations, respectively. Assuming that subunit interaction causes the cooperativity effect, the sets express the performance of soluble GDH's two subunits in two states of mutual interaction. From fitting the experimental data for several sugars with this equation, it appeared that their V1 values were similar, although their K1 values varied considerably. This showed that the cooperativity effect dramatically changes the performance of soluble GDH, as reflected by the V2 and K2 values for glucose (in phosphate buffer) being about 10-fold and 100-fold higher than the V1 and K1 values, respectively. Substituting the Ca2+ involved in activation of pyrroloquinoline quinone (PQQ) in soluble GDH by Sr2+ affected the cooperativity effect (an increase of the K1 value) but not the two turnover rates of the hybrid enzyme for glucose. The data suggest that the two catalytic cycles of soluble GDH have different rate-limiting steps compared with that of PQQ-containing methanol dehydrogenase.