Modification of Lys-6 and Lys-65 affects the structural stability of Taiwan cobra phospholipase A2.
To assess whether chemical modification of phospholipase A(2) (PLA(2)) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA(2) were selectively modified with trinitrobenzene sulfonate and pyridoxal-5'-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA(2) activity, but the Ca(2+)-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA(2) when thermal unfolding was conducted in the presence of 20 mM Ca(2+). Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca(2+). Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca(2+) pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA(2), our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA(2) activity.
Chang
Long-Sen
LS
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. lschang@mail.nsysu.edu.tw
Cheng
Yun-Ching
YC
Chen
Ching-Ping
CP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004546:Elapid Venoms / Q000201:enzymology*; D017815:Elapidae; D004795:Enzyme Stability; D054513:Group IV Phospholipases A2; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D011489:Protein Denaturation; D013624:Taiwan; D014302:Trinitrobenzenesulfonic Acid
D004546:Elapid Venoms; D014302:Trinitrobenzenesulfonic Acid; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D008239:Lysine