Günter Legler

Epoxyalkyl oligo-(1→4)-β-D-glucosides as active-site-directed inhibitors of cellulases

Abstract Eight alkyl oligo-β- D -glucosides having a terminal epoxide function have been synthesized and tested as active-site-directed, irreversible inhibitors for cellulases from an Oxyporus species, Aspergillus niger, and Asp. wentii. The D -glucosides showed very little reactivity, but increasing deactivation was observed with an increased number of D -glucose residues. Variation of chain length of the aglycon group showed a maximum of reactivity with 5 carbon atoms. This and the partial protection of the enzyme by cellobiose indicate that the deactivation is due to attack at the active site. The variation of the deactivation rate with pH is very similar to that of the cellulase activity, and, in each case, a carboxyl group seems to be involved as essential acid-catalyst. In the Oxyporus preparation, a cellobiosidase activity was detected with p-nitrophenyl β-cellobioside as substrate. This activity is due to a separate enzyme because it was not affected by (1,3/2,4)-5-cyclohexene-1,2,3,4-tetrol epoxide, which is a specific inhibitor of β- D -glucosidase, and showed only little reactivity with one of the cellulase inhibitors.

Composition, N-terminal amino acids, and chain length of a β-glucosidase from Aspergillus wenth

Abstract 1. 1.|The β-glucosidase A 3 (β- D -glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus wentii is an acidic glycoprotein of molecular weight 170 000 (G. LEGLER , Z. Physiol. Chem. , 348 (1967) 1359). Its isoelectric point was pH 3.80. Except for the preponderance of acidic amino acids and the presence of only 4 disulfide bonds, no unusual features were found in the amino acid composition. The protein contained about 21% carbohydrate, including 6.2% glucosamine. Mannose, and smaller amounts of glucose and fucose were identified as neutral sugars. 2. 2.|Determination of the N-terminal amino acids by the dansyl method gave 1 mole aspartic acid (or asparagine) per 170 000 g protein. 3. 3.|Attempts to dissociate the enzyme into subunits by reduction of the disulfide bonds, subsequent reaction with 5,5′-dithiobis-(2-nitrobenzoic acid), acrylonitrile, or ethyleneimine, and chromatography or electrophoresis in the presence of denaturing agents were unsuccessful. 4. 4.|The intrinsic viscosity of the reduced and denatured enzyme indicated a chain length between 910 and 1300 amino acid residues. 1200 residues are calculated from the amino acid analysis. 5. 5.|The molecular weight, determined by sedimentation analysis of the reduced and alkylated enzyme in 6 M guanidine hydrochloride, was 168 000 ± 5000. It is concluded that the enzyme consists of a single peptide chain of about 1200 amino acid residues. 6. 6.|ORD and CD measurements indicated that only about 15% of the molecule was present in a helical conformation. Conformational changes set in at 20° below pH 2.2 and above pH 7.5.

Die bestandteile des giftigen glykosidharzes aus Ipomoea fistulosa mart. et chois.

Abstract From the leaves of Ipomoea fistulosa (Convolvulaceae) a resinous high-molecular-weight ester glycoside has been isolated, similar to the resins found in the underground organs of other convolvulaceae. It consists of 7-hydroxy decanoic acid, 11-hydroxy tetradecanoic acid, 11-hydroxy hexadecanoic acid, and 3,11-dihydroxy tetradecanoic acid glycosidically bound to several residues of d -glucose, d -fucose, d -quinovose, and l -rhamnose to form glycosidic acids with an equivalent weight of 800 to 1300. A part of the 7-hydroxydecanoic acid was isolated as a β- d -quinovoside. The glycosidic acids are esterified with each other to form a mixture of closely related polyesters with an average molecular weight of about 25,000, showing a broad distribution curve in the ultracentrifuge. A part of the free hydroxyl groups is esterified with tiglic acid, with saturated C 2 , C 4 , and C 5 acids and with 3,11-dihydroxy tetradecanoic acid.