Roles of lysine-69 in dimerization and activity of Trimeresurus flavoviridis venom aspartate-49-phospholipase A2.
Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.
Nakamura
S
S
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
Nakai
M
M
Nakashima
K
K
Ogawa
T
T
Shimohigashi
Y
Y
Ohno
M
M
Kihara
H
H
Yamane
T
T
Ashida
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D016415:Sequence Alignment; D017841:Trimeresurus / Q000378:metabolism*; D014302:Trinitrobenzenesulfonic Acid / Q000378:metabolism / Q000494:pharmacology
D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; C029748:Trimeresurus venoms; D001224:Aspartic Acid; D014302:Trinitrobenzenesulfonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium