Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D.
The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D012276:Ricin / Q000032:analysis / Q000378:metabolism*; D012697:Serine Endopeptidases; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis* / Q000378:metabolism
D000596:Amino Acids; D010455:Peptides; D014364:Tryptophan; D012276:Ricin; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C036813:lysyl endopeptidase