Strong inhibitory effect of sugar-biphenylylboronic acid complexes on the hydrolytic activity of α-chymotrypsin

Boronic acids act as transition-state analogues for certain peptidases. The inhibitory effect of 2-, 3- and 4-biphenylylboronic acids (2a, 2b and 2c) on the hydrolytic activity of α-chymotrypsin has been investigated. These inhibitors were employed to monitor the binding event [formation of covalent bond with either serine residue (195) or histidine residue (57)] occurring in the active site by a fluorescence method. It was shown that the decrease in the fluorescence intensity, which is induced by the formation of a covalent bond with the boronic acid moiety, is well correlated with the inhibitory effect estimated by kinetic measurements. The inhibitory effect appeared in the order 2a < 2c ≪ 2b (K_i = 1.6 × 10^-6 mol dm ^-3). Interestingly, the inhibitory effect was further intensified by added saccharides. In particular, the combined system of 2b and D-glucose strongly inhibited the enzyme reaction, the inhibitory effect (K_i = 1.1 × 10^-7 mol dm^-3) being stronger than that of a specific inhibitor, chymostatin (K_i = 4.8 × 10^-7 mol dm^-3). Hence, saccharides act as a 'co-inhibitor' in the boronic acid inhibition system. This is a novel and efficient inhibition system for α-chymotrypsin (and probably more generally for other peptidases).