Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan, ROC.
Lin
S R
SR
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D004546:Elapid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D017815:Elapidae; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects*; D014302:Trinitrobenzenesulfonic Acid
D004546:Elapid Venoms; D014302:Trinitrobenzenesulfonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium