Bindings of Ca2+ and substrate analogs to a cobra venom phospholipase A2 in which the alpha-amino group is modified to an alpha-keto group.

Abstract

The pH dependence of the chemical reaction rate of p-bromophenacyl bromide (BPB) with His 48 of cobra (Naja naja atra) venom phospholipase A2, in which the alpha-NH2 group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 in the absence of Ca2+. The pH-dependence curve was monophasic with a midpoint at pH 7.9, which corresponds to the pK value of His 48 of the alpha-NH2-modified enzyme, whereas the curve for the intact enzyme was biphasic, indicating participation of two ionizable groups with pK values of 7.3 and 8.55 (Teshima et al. (1982) J. Biochem. 91, 1778-1788). These two groups were thus identified as His 48 and the alpha-NH2 group, respectively. The pH dependence of the binding constant of Ca2+ to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by measuring the tryptophyl fluorescence changes. The pH-dependence curve was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 13-20), and it was interpreted in terms of participation of His 48 and Asp 49 (pK 5.4). The absence of participation of the alpha-NH2 group in the Ca2+ binding was thus confirmed. Bindings of monodispersed n-dodecylphosphorylcholine (n-C12PC) and micellar n-hexadecylphosphorylcholine (n-C16PC) to the alpha-NH2-modified enzyme were studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) and tryptophyl fluorescence methods, respectively. The binding constant of the monodispersed substrate was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The binding constant of the micellar substrate to the modified enzyme in the presence of Ca2+ was also very similar to that for the intact enzyme-Ca2+ complex (Teshima et al. (1983) J. Biochem. 94, 223-232), and the pH-dependence curve was interpreted in terms of participation of His 48. On the other hand, the binding constant of the micellar substrate to the modified apoenzyme was much smaller than that for the intact apoenzyme. Nevertheless, the pH-dependence curve could be interpreted in terms of participation of His 48 and Asp 49. From these findings, it was concluded that the ionization state of the alpha-NH2 group of cobra venom phospholipase A2 is essentially irrelevant to the bindings of Ca2+ and also of the monodispersed and micellar substrates.