Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups.
The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.
Clarke
A J
AJ
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Ontario, Canada.
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives; D001487:Basidiomycota / Q000201:enzymology*; D002234:Carbodiimides / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D005944:Glucosamine / Q000494:pharmacology; D005959:Glucosidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012567:Schizophyllum / Q000201:enzymology*; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D002234:Carbodiimides; D002413:Cations, Divalent; D017485:1-Deoxynojirimycin; D005959:Glucosidases; D001617:beta-Glucosidase; D005944:Glucosamine; C013626:nojirimycin