The role of carboxyl groups in the function of endo-beta-1,4-glucanase from Schizophyllum commune.
The endo-beta-1,4-glucanase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed concanavalin A-Sepharose. The catalytic site of the enzyme has previously been proposed to consist of Glu-33 and Asp-50 that act in a manner analogous to lysozyme [Yaguchi, M., Roy, C., Rollin, C.F., Paice, M.G. & Jurasek, L. (1983) Biochem. Biophys. Res. Commun. 116, 408-411]. The role of carboxyl groups in the mechanism of action was delineated through kinetic and chemical modification studies. The rate of endoglucanase-catalysed hydrolysis of CM-cellulose was determined viscometrically at 30 degrees C, 0.06 M ionic strength and pH 2.5-9.0. The pH profile for maximum velocity gave the kinetic apparent pK values 3.8 and 6.6 and for initial velocity the pK values 3.7 and 6.1. Treatment of the endoglucanase with diethylpyrocarbonate resulted in the modification of all the four histidyl residues present in the enzyme with the retention of 95% of the original enzymatic activity. A water-soluble carbodiimide completely inactivated the cellulase and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. The pH dependence of the inactivation is consistent with the modification of carboxyl groups. The binding of an inhibitor, cellobiose, prior to carbodiimide modification protected the enzyme from rapid inactivation. Titration of the enzyme with dithiobis(2-nitrobenzoic acid) indicated the absence of free thiol groups. Reaction of the endoglucanase with tetranitromethane resulted in the modification of six of the fourteen tyrosyl residues of the enzyme with approximately 35% diminution in activity.
D000363:Agaricales / Q000201:enzymology*; D002266:Carboxymethylcellulose Sodium; D002384:Catalysis; D002475:Cellobiose / Q000494:pharmacology; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D012567:Schizophyllum / Q000201:enzymology*
D002475:Cellobiose; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium