Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis.
Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Teshima
K
K
Department of Hygienic Chemistry, Faculty of Pharmacy, Hoshi University, Tokyo.
Ikeda
K
K
Miyake
T
T
Imamura
M
M
Inoue
S
S
Samejima
Y
Y
Kawauchi
S
S
Omori-Satoh
T
T
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008823:Micelles; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002021:Buffers; D003435:Crotalid Venoms; D008823:Micelles; D010713:Phosphatidylcholines; C029748:Trimeresurus venoms; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium