Ca2+-assisted, direct hydride transfer, and rate-determining tautomerization of C5-reduced PQQ to PQQH(2), in the oxidation of beta-D-glucose by soluble, quinoprotein glucose dehydrogenase

Spectral and kinetic studies were performed on enzyme forms of soluble gluc ose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in wh ich the PQQ-activating Ca2+ was absent (Holo X) or was replaced with Ba2+ ( Ba-E) or in which PQQ was replaced with an analogue or a derivative called "nitroPQQ" (E-NPQ). Although exhibiting diminished rates, just like sGDH, a ll enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron a cceptor. On inspection of the plots for the reductive half-reaction, it app eared that the enzyme forms exhibited a negative cooperativity effect simil ar to that of sGDH itself under turnover conditions, supporting the view th at simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E w ith glucose showed a fluorescing transient previously observed in the react ion of sGDH with glucose-1-d, whereas no intermediate was detected at all i n the reactions of E-NPQ and Hole X. Using hydrazine as a probe, the fluore scing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Hole X, but E-NPQ did not react with hydrazine. When this is combined with other pr operties of E-NPQ and the behavior of enzyme forms containing a PQQ analogu e, we concluded that the catalytic potential of the cofactor in the enzyme is not determined by its adduct-forming ability but by whether it is or can be activated with Ca2+, activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the red uced cofactor in the enzyme. This conclusion, together with the observed de uterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and t hat already known on transient decay, indicate that the sequential steps in the mechanism of sGDH must be (1) reversible substrate binding, (2) direct transfer of a hydride ion (reversible or irreversible) from the C1 positio n of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-de termining tautomerization of the fluorescing, CS-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxid ation of PQQH(2) by an electron acceptor. The PQQ-activating Ca2+ greatly f acilitates the reactions occurring in step 2. His144 may also play a role i n this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity o f the proposed mechanism is discussed for other PQQ-containing dehydrogenas es.