INFLUENCE OF PH ON THE KINETIC AND SPECTRAL PROPERTIES OF PHOSPHOLIPASE-A2 FROM BITIS-GABONICA (GABOON ADDER) SNAKE-VENOM

The influence of pH on the kinetic parameters kcat and kcat Kb, and the spectral properties of Bitis gabonica phospholipase A2, showed that the activity of the enzyme is controlled by groups of pK values 6·8 and 9·2. These groups were found to exhibit enthalpies of ionization (ΔHion) of 7·1 Kcal/mole and 6·1 Kcal/mole respectively which are characteristic of ΔHion of histidine and tyrosine residues. While the dissociation constant (Kia) for reaction between enzyme and Ca2+ was affected by pH, the Michaelis constant (Kb) of the enzyme for the phospholipid substrate was found to be uninfluenced in the pH range tested i.e. pH 5·-9·9.0. The binding of Ca2+ to the enzyme is inhibited by protonation of a group ofpK ca. 6·0. The ΔHion of - 1·6 Kcal/mole calculated for this group is in the range of the values found for carboxyl groups. The various functions of nucleophile, proton donor and Ca2+ binding site may be assigned to the side chains of structurally invariant residues which for the B. gabonica phospholipase A2 are located at His-45, Tyr-25 and Asp-46 respectively. Non-competitive inhibition of the H+ was observed with respect to both Ca2+ and lecithin which probably involves an effect of the proton on free enzyme and the interconversion of the central complexes. © 1979.