Structural characterization of a glycoprotein cellulase, 1,4-beta-D-glucan cellobiohydrolase C from Trichoderma viride.
A glycoprotein enzyme, 1,4-beta-D-glucan cellobiohycrolase (EC 3.2.1.91) form C, was purified to electrophoretic homogeneity by a procedure which permitted isolation of gram quantities from a commercial Trichoderma viride culture filtrate preparation. Purified cellobiohydrolase C has an E1%/280 nm = 14.2 and degrades both microcrystalline and phosphoric acid-swollen cellulose to cellobiose. The cellobiohydrolase C contains 26.4, 4.8, 2.4 and 3.4 mol of mannose, glucose, galactose and glucosamine, respectively, per mol of enzyme (molecular weight, 48 400). Methylation analysis of cellobiohydrolase glycopeptides indicates an average carbohydrate chain length of two residues. Alkaline borohydride treatment of cellobiohydrolase C released neutral carbohydrate which is bound through an average of 16.7 O-glycosidic linkages to serine and threonine per molecule of enzyme. Glucosamine was not released from the protein by alkaline treatment. Analysis of alkaline borohydride-released carbohydrate by high pressure liquid chromatography demonstrated that an average enzyme molecule contains 8.8 mono-, 1.8 di-, 4.6 tri-, 1.2 tetra-, and 0.4 pentasaccharide chains. The linkages between the neutral monosaccharides are (1 leads to 6) as shown by gas chromatography - mass spectrometry of partially methylated residues. The (1 leads to 6) linkage is consistent with the stability of the linkages to alkaline conditions and the destruction of all neutral carbohydrate by periodate. Action of alpha-mannosidase indicates that some oligosaccharide chains contain alpha-mannose as the terminal residue.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005944:Glucosamine / Q000032:analysis; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D006601:Hexoses / Q000032:analysis; D003904:Mitosporic Fungi / Q000201:enzymology*; D011485:Protein Binding; D014242:Trichoderma / Q000201:enzymology*
D000596:Amino Acids; D006023:Glycoproteins; D006601:Hexoses; D006026:Glycoside Hydrolases; D005944:Glucosamine