Participation of an ionizable group with pK 8.55 in the reaction of p-bromophenacyl bromide with His 48 of cobra venom phospholipases A2.

Abstract

The chemical reaction of p-bromophenacyl bromide (BPB) with His 48 of cobra venom phospholipases A2 (N. naja siamensis, N. naja kaouthia, and N. naja atra) was studied at 25 degrees C and ionic strength 0.1 by following the decreases in the fluorescence intensity of 8-anilino-1-naphthalene sulfonate (ANS) and/or in the enzymatic activity. The effect of the BPB concentration on the pseudo-first-order rate constant, chi obs, was studied for the N. naja atra enzyme and the dissociation constants of a noncovalent intermediate were determined to be 5.6 x 10(-4) and 1.8 x 10(-4) M at pH 8.4 and 9.3, respectively. The ph-dependence curve of chi obs, obtained at fixed concentration(s) of BPB was found to be biphasic for all three enzymes. The analysis showed that two ionizable groups with pK values of 7.3 and 8.55 participated in this reaction. The reaction of BPB with the modified enzyme of N. naja atra lacking the N-terminal octapeptide, which had been prepared by the CNBr-cleavage method, was followed by measuring the decrease in the tryptophyl fluorescence. Since this modified enzyme showed a monophasic pH-dependence curve lacking the latter transition, the former group was assigned to His 48 and the latter to the alpha-amino group located nearby in the active site. The pK value of His 48 determined at present was in good agreement with that estimated from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20). The pK value of the alpha-amino group of this enzyme, 8.55, was found to be somewhat higher than that of the A. halys blomhoffii enzyme, 7.3 (Ikeda & Samejima (1981) J. Biochem. 90, 799-804 and Ikeda et al (1981) J. Biochem. 90, 1125-1130), but quite similar to that for the porcine pancreatic enzyme, 8.4 (van Dam-Mieras et al. (1976) Nobel Symp. 34, 177-197 and Slotboom et al. (1978) Biochemistry 17, 4593-4600).