Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait).
The N-terminal alpha-amino groups of beta 1-bungarotoxin (beta 1-Bgt) from Bungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the alpha-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between beta 1-Bgt and 8-anilinonaphthalene sulfonate (ANS) and revealed that beta 1-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca2+ and ANS, indicating that the N-terminal region is not involved in Ca2+ and substrate binding. A fluorescence study revealed that the alpha-amino group of the A chain was in the vicinity of substrate binding site and that the TNP alpha-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for beta 1-Bgt.
Chang
L S
LS
Institute of Life Sciences, National Tsing Hua University, Taiwan, Republic of China.
Yang
C C
CC
D000596:Amino Acids / Q000032:analysis; D002038:Bungarotoxins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014301:Trinitrobenzenes
D000596:Amino Acids; D002038:Bungarotoxins; D007202:Indicators and Reagents; D014301:Trinitrobenzenes; D003488:Cyanogen Bromide; D002118:Calcium