Glucosylceramidase from calf spleen. Characterization of its active site with 4-n-alkylumbelliferyl beta-glucosides and N-alkyl derivatives of 1-deoxynojirimycin
| Autor: | Günter Legler, Harald Liedtke |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
1-Deoxynojirimycin
Hot Temperature Chemical Phenomena Detergents Protonation Biochemistry Micelle Medicinal chemistry Substrate Specificity chemistry.chemical_compound Glucosides Animals Carboxylate Glycosides Umbelliferones Binding site Alkyl chemistry.chemical_classification Glucosamine Binding Sites biology Chemistry Active site Substrate (chemistry) Kinetics Ionic strength biology.protein Glucosylceramidase Cattle Glucosidases Spleen |
| Zdroj: | Biological chemistry Hoppe-Seyler. 366(12) |
| ISSN: | 0177-3593 |
| Popis: | The beta-glucosides of 4-heptyl-, -nonyl-, and -undecylumbelliferone were synthesized and their substrate properties studied with calf spleen glucosylceramidase. Self-association of the free long chain alkylumbelliferones in aqueous buffer was inferred from their low fluorescence in the absence and strongly enhanced fluorescence in the presence of detergents. Association of the higher alkylumbelliferyl glucosides with detergent micelles was indicated by the influence of detergent on solubility and on enzyme activity which differed markedly between the methyl and the higher alkyl substrates. Compared to 4-methylumbelliferyl beta-glucoside their Km was 14 to 23 times smaller and Vmax/Km 20 to 30 times larger with no significant difference between the nonyl and undecyl derivatives. The enzyme was inhibited by 1-deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol, dNM) and a series of its N-alkyl derivatives with Ki-values that ranged from 390 microM for the parent compound to 330 microM for the butyl derivative and 0.08 microM for the tetradecyl derivative. The biphasic linear plot of - RT X 1n [Ki/Ki (dNM)] vs. chain length is interpreted in terms of an aglycon binding site that has an extended hydrophobic region starting at about 5 carbon atoms from the catalytic site. dNM inhibited greater than or equal to 10(3) times better than D-glucose, and N-decanoyl-dNM was a very weak inhibitor compared to N-decyl-dNM. It is concluded that the formation of an ion pair consisting of the protonated dNM derivative and an essential carboxylate at the catalytic site makes a large contribution to the binding energy. Strong shielding of this site from the aqueous environment is indicated by identical effects of ionic strength on Km and Ki. |
| Databáze: | OpenAIRE |
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