Isotope Effects and Temperature Dependences in the Action of the Glucose Dehydrogenase of the Mesophilic Bacterium Bacillus megaterium

Abstract

The glucose dehydrogenase of the mesophilic bacterium Bacillus megaterium (optimal growth around 35 °C) exhibits non-linear Eyring temperature dependences from 25 to 55 °C in its catalysis of the oxidation by hydride-transfer to NAD+ of the β-anomers of 1-h-D-glucose and 1-d-D-glucose (rate constant kcat/KMβ). A break around 300K separates a high-T region from a low-T region. In the high-T region, isotopic enthalpies of activation within a considerable experimental error are equal to zero. In the low-T region, the enthalpies of activation are roughly equal for the isotopic substrates but are different from zero. An alternative treatment with Eyring plots taken as effectively linear produces enthalpies of activation having the unusual feature of being larger for the H-substrate (26 kJ/mol) than for the D-substrate (21 kJ/mol). Compensation of the enthalpic effect by a more positive entropy for the H-substrate then reproduces the isotope effects. For oxidation by NADP+ of the same pair of isotopic glucose substrates, catalysis by the glucose dehydrogenase of Thermoplasma acidophilum, a thermophilic archaeon, leads to temperature dependences characterized by a high-T region and a low-T region separated by a gentle thermal transition (K. Anandarajah, K.B. Schowen, and R.L. Schowen, Z. phys. Chem. 2008, 222, 1333–1347). Tentative approaches to a mechanistic interpretation of both cases rely on models featuring configurational searches of the enzyme for tunneling states, followed by hydrogen-transfer tunneling, although explanations can be constructed also on the basis of simple transition-state stabilization without tunnelling.