Chemical Modification of the Histidine Residue in Phospholipase A, (Naja naja naja)

Reaction of phospholipase A, (Naja naja naja) with pbromophenacyl bromide leads to almost complete loss of enzymatic activity. The rate of inactivation is pa-dependent with ,pK, = 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidinelmol of enzyme as judged by amino acid analysis and incorporation studies with W-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase AZ, by acylation of two amino groups, a tyrosine, and 0.5 mol of histidinelmol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidinelmol of enzyme, can be further inactivated by treatment withp-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the Wlabeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A, with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.