Spectral perturbations of the histidine and tryptophan in cobra venom phospholipase A2 upon metal ion and mixed micelle binding.

Metal ions and nonionic surfactantlphospholipid mixed micelles are shown to affect the structure of pure cobra venom phospholipase A, (Nuja naju Baja). This small phospholipase, with 1 histidine and 1 tryptophan residue per molecule, has an absolute requirement for Ca’+, although many divalent metal ions can bind to it. Inhibitory metals include Zn2+, Ba’+, and Sr’+, whereas MnZ+, Mg2+, and Cd’-+ bind without altering enzyme activity. The pH rate profile of the reaction with phosphatidylcholine as substrate shows that Caz+ lowers the apparent p& controlling the reaction (for 10 m,n Ca’+, p& = 5.6; for 0.9 rnx Ca2+, p& = 6.3). The binding of Ca2+ to the enzyme is pa-dependent with pK, = 5.9 and Kc,,+ = 0.15 mM for the unprotonated form of the enzyme as determined by both fluorescence emission and UV difference spectroscopy. The Ca2+-induced difference spectra are characterized by a blue-shifted tryptophan perturbation at basic pH and a red-shifted tryptophan perturbation at acidic pH. The difference spectrum induced by Cal+ at acid pH is similar to the titration difference spectrum observed with apoenzyme. The latter shows a pH dependence with a pK,, = 6.8 to 7.1. Gel filtration studies of the molecular weight of the enzyme suggest that at acidic pH and high enzyme concentrations, this conformational change involves dimerization of the enzyme. These results suggest that when Ca’+ binds to phospholipase A,, it triggers a conformational change which lowers the pK,, of a critical residue, probably the histidine, and in so doing alters somewhat the monomer-dimer equilibrium of the enzyme. ‘H nmr studies of the aromatic region of the enzyme are consistent with this interpretation. Furthermore, binding studies with substrate in mixed mieelles and metal ions suggest that the reaction of phospholipase A, is ordered; enzyme must bind metal ion before it can bind to phospholipid in mixed micelles.