Influence of pH on the kinetic and spectral properties of phospholipase A 2 from Bitis gabonica (Gaboon adder) snake venom

Abstract

The influence of pH on the kinetic parameters k cat and k cat K b , and the spectral properties of Bitis gabonica phospholipase A 2, showed that the activity of the enzyme is controlled by groups of p K values 6·8 and 9·2. These groups were found to exhibit enthalpies of ionization (ΔH ion) of 7·1 Kcal/mole and 6·1 Kcal/mole respectively which are characteristic of ΔH ion of histidine and tyrosine residues. While the dissociation constant ( K ia) for reaction between enzyme and Ca 2+ was affected by pH, the Michaelis constant ( K b) 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 Ca 2+ to the enzyme is inhibited by protonation of a group ofp K ca. 6·0. The ΔH ion 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 Ca 2+ binding site may be assigned to the side chains of structurally invariant residues which for the B. gabonica phospholipase A 2 are located at His-45, Tyr-25 and Asp-46 respectively. Non-competitive inhibition of the H + was observed with respect to both Ca 2+ and lecithin which probably involves an effect of the proton on free enzyme and the interconversion of the central complexes.