Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives.
A beta-glucosidase/beta-galactosidase with Mr 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 Km or Ki of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower Vmax than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for kcat/Km of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with Ki (= Km) 2.2 microM and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl beta-glucoside. The product sphingosine was inhibitory with Ki 0.30 microM. 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 CH2 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 approximately 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 beta-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.
D017485:1-Deoxynojirimycin; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005959:Glucosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011609:Psychosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D017485:1-Deoxynojirimycin; D011609:Psychosine; C035742:sphingosyl beta-glucoside; D005959:Glucosidases; D001617:beta-Glucosidase; D005944:Glucosamine