RESUMO
The contribution of the hydroxyl groups at C-2 and C-4 and of the hydroxy-methyl group at C-5 of beta-glucopyranosides to their hydrolysis by beta-glucosidase A3 (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus wentii was investigated with 4-methylumbelliferyl-beta-glucosides with appropriate structural modifications. Relative hydrolysis rates expressed by kcat/kcat (glucoside) are: 2-deoxy, 4. 10(-6); 2-deoxy-2-amino, 2.4 . 10(-4); 2-deoxy-2-ammonio, less than 1 . 10(-6); 4-deoxy, 1.8 . 10(-4); xyloside, 6.3 . 10(4); galactoside, less than 1 . 10(-6). Binding to the active site as measured by the Km value of these substrates or by the Ki value of the appropriate inhibitors is only moderately decreased by the above modifications. A temperature study with the 2-deoxyglucoside showed that the decrease in kcat is not due to a change in delta H but to a more negative delta S. The steady-state hydrolysis of the 2-deoxyglucoside is approached with a "burst" (rate constant 0.13 min-1) at pH 6 and 1 mM substrate; deglycosylation of the enzyme is partially rate-limiting. Rate constants for glycosylation and deglycosylation calculated from pre-steady-state kinetics were in good agreement with the constants calculated from experiments where the 2-deoxyglucoside was used as an inhibitor for the hydrolysis of the glucoside and where a slow approach to the steady state of the inhibited reaction is observed.
