Role of Ca2+ in the Substrate Binding and Catalytic Functions of Snake Venom Phospholipases A2

Phospholipases A2 are classsfied into two groups, I and II, according to differences in the polypeptide-chain length and the intramolecular-disulfide bondings. The effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed and mi cellar phosphatidyl-cholines, catalyzed by a cobra (Naja naja atra) enzyme (Group I) and by mamushi {Agkistrodon halys blomhoMi) and habu (Trimeresurus ftavoviridis) enzymes (Group II), were studied by the pH-stat assay method at 25°C, pH 8.0–8.2, and ionic strength 0.1–0.2. The results were compared with those reported for other Group I and II enzymes. The Ca2+ binding was clearly shown to be essential for the catalysis of all the phospholipases A2. However, the substrate binding to Group I enzymes was found to be independent of the Ca2+ binding. On the other hand, the substrate binding to Group II enzymes was facilitated more than 10 times by the binding of Ca2+ to the enzymes. This was interpreted in terms of conformation changes of the peptide loop of residues 26 to 44 accompanying the Ca2+ binding. The latter result, but not the former, seems compatible with the hypothesis for interpreting the catalytic mechanism of phospholipases A2 that an intermediate complex should be stabilized by the coordination of the bound Ca2+ ion with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743–750 and (1981) Rev. Physiol Biochenu Pharmacol, 91, 91–203]. According to the similarity in the primary and tertiary structures of the active sites of both types of enzymes [Reneteeder et al. (1985) J. Biol. Chem 260, 11627–11634], it is supposed that similar intermediate complexes may occur even for Group I enzymes, at least in the transition state of the productive complexes.