1989 Volume 105 Issue 4 Pages 565-572
The phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25°C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1, 2-bis [4-(1-pyreno) butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal α-NH2 group had been selectively converted into an α-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the α-NH2 modified enzymes were also studied at 25°C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the α-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53). The enzyme dimerization was found to reduce the binding constant of Ca2+ to the enzyme at neutral and acidic pH values, suggesting that the way in which one enzyme molecule is brought into contact with the other is unfavorable to the Ca2+ binding. This result seems compatible with the recent X-ray crystallographic data on the dimeric phospholipase A2 of Crotalus atrox (Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749), which belongs to the same family, Crotalidae, as T. flavoviridis.
