External yeast beta-fructosidase: stereospecific labeling by [3H]conduritol-beta-epoxide and isolation of labeled peptides from the active site.
1. External yeast beta-fructosidase (beta-D-fructofuranoside fructohydrolase EC 3.2.1.26) was labeled by [3H]conduritol-B-epoxide, an active-site directed inhibitor of this enzyme. During the inactivation 1--2 mol of inhibitor were bound covalently per mol of enzyme. The labeled enzyme was digested with pepsin and 3H-labeled peptides were isolated. 2. Conduritol-B-epoxide is a racemic mixture of 1-D-1,2-anhydro-myo-inositol and 1-L-1,2-anhydro-myo-inositol, but only the latter one was now shown to be the reactive component. 3. The label was released from the enzyme by milk alkaline treatment and identified by paper chromatography as 1-D- or L-chiro-inositol, which suggested that an ester linkage had been formed by reaction of the epoxide with a carboxylate group at the active site of the enzyme. Position and role of this carboxylate group are discussed.
D000345:Affinity Labels; D001665:Binding Sites; D007294:Inositol / Q000031:analogs & derivatives*; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000302:isolation & purification; D013393:Sucrase / Q000378:metabolism*; D014316:Tritium
D000345:Affinity Labels; D010446:Peptide Fragments; D014316:Tritium; D007294:Inositol; D013393:Sucrase