Isolation of a cytosolic β-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives

Abstract

A β-glucosidase/β-galactosidase with M r 52,500 was isolated from calf liver cytosol by a four-step procedure incorporating affinity chromatography on N-(9-carboxynonyl)-deoxynojirimycin-AH-Sepharose. Its pH optimum was at 5.8 with half-maximal activity at pH 3.5 and 8.6. Affinity for gluco compounds expressed by K m or K i , of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower V max than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for k cat K m of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with K i (= K m) 2.2 μ m and hydrolyzed with a V max that was 50-fold lower than the V max for 4-methylumbelliferyl β-glucoside. The product sphingosine was inhibitory with K i 0.30 μM. A systematic study with alkyl glucosides and glucosylamines defined the aglycon site as a narrow, strongly hydrophobic cleft able to accommodate up to 10 methylene groups. Each CH 2 group contributed 3.1 kJ/mol to the standard free energy of binding. The inhibition by gluco-and galactosylamine and by 1-deoxynojirimycin and its D- galacto analog was ~200-fold better than by corresponding nonbasic compounds. pH dependence of the inhibition and comparison with permanently cationic glycosyl derivatives showed that the nonprotonated form was the inhibiting species. This feature puts the cytosolic β-glucosidase in the large class of glycoside hydrolases which strongly bind basic glycosyl derivatives by their protonation at the active site and formation of a shielded ion pair with the carboxylate of an aspartic or glutamic side chain.