β-glucosidase from the hyperthermophilic T. maritima is a retaining family 1 glycohydrolase. The action of this enzyme has been examined against a series of synthesized phenyl glucosides to construct Brønsted plots. The plot of kcat vs pKa exhibits a concave down shape suggesting a change in the rate limiting step from glycosylation to de-glycosylation. Aglycones with pKa<8 show little dependence on leaving group ability, while the poorer substrates for kcat (βlg=-0.39) and all substrates for kcat/KM (βlg=-0.41) were found to depend on leaving group ability to a similar extent. This is consistent with glycosylation being the committed step in catalysis. Solvent kinetic isotope effect studies showed that activity on para nitrophenyl glucoside (pKa=7.15) was unaffected while phenyl glucoside (pKa=9.95) was cleaved more slowly in deuterium oxide (kL/kH=1.54). Solvent kinetic isotope effects saw little change between measurement at 25 and 60 °C. This work suggests a dissociative glycosylation transition state that is stabilized in part by proton donation and a de-glycosylation transition state that involves no proton transfer.
