Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F.
Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-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 beta-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 beta-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 beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.
D000818:Animals; D002236:Carbohydrate Conformation; D002417:Cattle; D053138:Cyclohexenes; D003600:Cytosol / Q000201:enzymology*; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006868:Hydrolysis; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors*
D053138:Cyclohexenes; C036388:6-bromoconduritol; D007294:Inositol; D005959:Glucosidases; D001617:beta-Glucosidase