Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.
Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.
Wacker
H
H
Department of Biochemistry, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule-Zentrum, Zürch.
Keller
P
P
Falchetto
R
R
Legler
G
G
Semenza
G
G
D017485:1-Deoxynojirimycin; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004247:DNA; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D007422:Intestines / Q000201:enzymology*; D043322:Lactase; D010694:Lactase-Phlorizin Hydrolase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D009834:Oligo-1,6-Glucosidase / Q000378:metabolism*; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D013393:Sucrase / Q000378:metabolism*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D017485:1-Deoxynojirimycin; D007294:Inositol; D004247:DNA; D006026:Glycoside Hydrolases; D009834:Oligo-1,6-Glucosidase; D043322:Lactase; D001616:beta-Galactosidase; D013393:Sucrase; D010694:Lactase-Phlorizin Hydrolase; D005944:Glucosamine; C011423:conduritol epoxide