Hydrolysis of p-nitrophenyl-β-d-glucoside by cytosolic β-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow ( Ki(max) Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The β-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as ( 1,3,4 2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal β-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic β-glucosidase was caused by bromoconduritol F (( 1,2,4 3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with Ki(max) Ki 105 M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the β-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t 1 2 ~20 min.
ASJC Scopus subject areas
- Molecular Biology