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

Günter Legler, Erhard Bieberich

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37 Scopus citations

Abstract

A β-glucosidase/β-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 μm and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl β-glucoside. The product sphingosine was inhibitory with Ki 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 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 ~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.

Original languageEnglish (US)
Pages (from-to)427-436
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume260
Issue number1
DOIs
StatePublished - Jan 1988

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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