Mechanism of Agrobacterium beta-glucosidase: kinetic analysis of the role of noncovalent enzyme/substrate interactions.

The role of noncovalent interactions in the catalytic mechanism of the Agrobacterium faecalis beta-glucosidase was investigated by steady-state and pre-steady state kinetic analysis of the hydrolysis of a series of monosubstituted aryl glycosides, in which the hydroxyl groups on the glycone were substituted by hydrogen or fluorine. Contributions of each hydroxyl group to binding of these substrates at the ground state are relatively weak (interaction energies of 3.3 kJ/mol or smaller) but are much greater at the two transition states (glycosylation and deglycosylation). The strongest transition state interactions were at the 2 position (at least 18 and 22 kJ/mol for glycosylation and deglycosylation, respectively) with the interactions at the 3 and 6 positions contributing at least another 9 kJ/mol of binding energy at both transition states. The interaction at the 4 position is less crucial to transition state binding but important for stabilization of the glycosyl-enzyme intermediate. Comparison of observed rates with those for spontaneous hydrolysis of the same substrates provides evidence for oxocarbenium ion character at both transition states, that for deglycosylation apparently having the greater positive charge development at the anomeric center.