Glucosylceramidase from Calf Spleen. Characterization of its Active Site with 4-n-Alkylumbelliferyl (β-glucosides andN-Alkyl Derivatives of 1-Deoxynojirimycin

Abstract

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 signficant 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 .mu.M for the parent compound to 330 .mu.M for the butyl derivative and 0.08 .mu.M for the tetradecyl derivative. The biphasic linear plot of -RT .times. 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 .gtoreq. 103 times better than D-glucose, and N-decanoyld-NM 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 indentical effects of ionic strength on Km and Ki.