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 subclass of class I (class IL; corresponds to class IV) chitinase. The positions of disulfide bonds in this chitinase were examined. Chitinase protein was digested with acid protease and thermolysin, and the resulting disulfide bond containing peptides were separated by reversed-phase HPLC and detected using the SBD-F (7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt) method. Four intradisulfide bonds containing peptides were purified and three disulfide bonds in the catalytic domain were identified as Cys-66 and Cys-115, Cys-128 and Cys-136, and Cys-218 and Cys-250. Location of disulfide bonds in the catalytic domain was identical to that of barley class II chitinase but different from 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 class II chitinases strongly suggests that this region is important for formation of the active site.
Araki
T
T
Department of Biochemistry, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto, Japan. taraki@ktmail.ktokai-u.ac.jp.
Kuramoto
M
M
Torikata
T
T
D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry*; D003553:Cystine / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013439:Sulfhydryl Reagents; D014675:Vegetables / Q000201:enzymology*
D004220:Disulfides; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D007527:Isoenzymes; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D003553:Cystine; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D002688:Chitinases