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Query: EC:3.2.1.21 (beta-glucosidase)
 3,280 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives. 296 89

Glucosylsphingosine is a potent inhibitor of lysosomal glucocerebrosidase and the broad-specificity, cytosolic beta-glucosidase of human liver. In the present study, it was demonstrated that the broad-specificity beta-glucosidase was also inhibited by galactosylsphingosine. The inhibition was observed when the enzyme was assayed for beta-glucosidase, beta-galactosidase, beta-xylosidase, and alpha-arabinosidase activities. Inhibition was of the mixed-type and the degree of inhibition depended on the substrate. For example, galactosylsphingosine was a more potent inhibitor of beta-glucosidase activity (I0.5 = 0.3 mM) than beta-xylosidase activity (I0.5 = 1.2 mM). In addition, the observation that the broad-specificity, cytosolic beta-glucosidase was inhibited by hydrophobic glycosphingolipids prompted the definition of a revised purification procedure which took advantage of hydrophobic affinity chromatography. This revised purification scheme employed Octyl-Sepharose and yielded the largest (68,000 Da) and most active (470,000 nmol h-1 mg protein-1) beta-glucosidase preparation yet described. The beta-glucosidase preparation contained 19% serine and 17% glycine, while 24% of the total amino acids were hydrophobic. The results of this study document the presence of a sphingolipid binding site on the broad-specificity beta-glucosidase. The implications of galactosylsphingosine inhibition of cytosolic beta-glucosidase and the possible role of the enzyme in glycosphingolipid metabolism are discussed.
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PMID:Galactosylsphingosine inhibition of the broad-specificity cytosolic beta-glucosidase of human liver. 391

Glucosylsphingosine has been isolated for the first time as a natural constituent from Gaucher's spleen. On thin-layer chromatography, it migrates with authentic glucosylsphingosine, yielding a positive color reaction with ninhydrin for the amino group and with alpha-naphthol-sulfuric acid for the carbohydrate residue. N-Acylation with palmitic acid gave rise to glucosylceramide, which was cleaved by purified glucosylceramide: beta-glucosidase to ceramide. Gas-liquid chromatography of the trimethylsilyl derivative showed a retention time similar to authentic glucosylsphingosine. Gas-liquid chromatographic analysis of the trimethylsilyl derivatives after methanolysis revealed the presence of only glucose and C(18)-sphingosine. Mass spectral data further supported the structural identity with glucosylsphingosine.
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PMID:Isolation and characterization of glucosylsphingosine from Gaucher's spleen. 441 1

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.
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PMID:Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites. 642 91