Ca2+-Assisted, Direct Hydride Transfer, and Rate-Determining Tautomerization of C5-Reduced PQQ to PQQH2, in the Oxidation of β-d-Glucose by Soluble, Quinoprotein Glucose Dehydrogenase

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Abstract
Spectral and kinetic studies were performed on enzyme forms of soluble glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in which the PQQ-activating Ca(2+) was absent (Holo X) or was replaced with Ba(2+) (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, all enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron acceptor. On inspection of the plots for the reductive half-reaction, it appeared that the enzyme forms exhibited a negative cooperativity effect similar to that of sGDH itself under turnover conditions, supporting the view that simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E with glucose showed a fluorescing transient previously observed in the reaction of sGDH with glucose-1-d, whereas no intermediate was detected at all in the reactions of E-NPQ and Holo X. Using hydrazine as a probe, the fluorescing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Holo X, but E-NPQ did not react with hydrazine. When this is combined with other properties of E-NPQ and the behavior of enzyme forms containing a PQQ analogue, 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 Ca(2+), activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the reduced cofactor in the enzyme. This conclusion, together with the observed deuterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and that 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 position of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-determining tautomerization of the fluorescing, C5-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxidation of PQQH(2) by an electron acceptor. The PQQ-activating Ca(2+) greatly facilitates the reactions occurring in step 2. His144 may also play a role in this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity of the proposed mechanism is discussed for other PQQ-containing dehydrogenases.