POSITIONS OF DISULFIDE BONDS IN YAM (DIOSCOREA-JAPONICA) ACIDIC CLASS-IL (CLASS-IV) CHITINASE

Yam acidic class I chitinase belongs to a low molecular weight subclas s of class I (class IL; corresponds to class IV) chitinase. The positi ons of disulfide bonds in this chitinase were examined, Chitinase prot ein was digested with acid protease and thermolysin, and the resulting disulfide bond containing peptides were separated by reversed-phase H PLC and detected using the SBD-F (7-fluorobenzo-2-oxa-1,3-diazole-4-su lfonic acid ammonium salt) method. Four intradisulfide bonds containin g peptides were purified and three disulfide bonds in the catalytic do main were identified as Cys-66 and Cys-115, Cys-128 and Cys-136, and C ys-218 and Cys-250. Location of disulfide bonds in the catalytic domai n was identical to that of barley class II chitinase but different fro m rye class II chitinase at the C-terminal. Conservation of S-S bonds at the N-terminal half of the catalytic domain between class I and cla ss II chitinases strongly suggests that this region is important for f ormation of the active site. (C) 1996 Academic Press, Inc.