Binding specificity and reactivity studies on a broad-specificity beta-glycosidase from porcine kidney.
A broad-specificity beta-glycosidase from porcine kidney was purified to homogeneity. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis showed that it had a monomeric molecular weight of 55,000-60,000. Gel filtration showed native molecular weight of about 115,000. These data imply that the native enzyme is a dimer. The enzyme can catalyze the hydrolysis of beta bonds between glycosides and 4-methylumbelliferone or nitrophenol yielding D-fucopyranose, D-galactopyranose, D-glucopyranose, D-xylopyranose, and D-mannopyranose and of alpha bonds to yield L-arabinopyranose. This is the first study that shows a mammalian broad-specificity cytosolic beta-glycosidase carrying out a reaction with a beta-D-mannopyranoside. The nature of the broad specificity was studied with inhibitors. Similar inhibitor constants were found regardless of whether the substrate was a beta-D-glucopyranoside or a beta-D-galactopyranoside, so the enzyme probably has only one binding site with a broad specificity. The enzyme prefers to bind compounds with an axial hydroxyl at the 2 position and an equatorial hydroxyl at the 4 position; the 3 position does not affect binding significantly. The hydroxyl at the 6 position affects binding, but binding at that position depends on the configurations at the 2 and 4 positions. Thus, there must be some interactions between these three positions (2, 4, and 6). Lactones are also good inhibitors and this may relate to strain effects.
Huber
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RE
Division of Biochemistry, Faculty of Science, University of Calgary, Alta., Canada.
Brockbank
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RL
D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008358:Mannose / Q000494:pharmacology; D008970:Molecular Weight; D010429:Pentoses / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
C030561:4-methylumbelliferyl glycosides; D006027:Glycosides; D046911:Macromolecular Substances; D010429:Pentoses; D006923:Hymecromone; C043333:lyxose; D006026:Glycoside Hydrolases; D005947:Glucose; D008358:Mannose; D005690:Galactose