Added NaCl or anionic amphiphiles increase the rate of hydrolysis of dispersions of zwitterionic phospholipid by pancreatic phospholipase A2 (PLA2). Two effects of the negative charge at the interface have been dissected: enhanced binding of the enzyme to the interface, and k*cat activation of the enzyme at the interface [Berg et al. (1997) Biochemistry 36, 14512-14530]. Results reported here show that the structural basis for the k*cat activation is predominantly through cationic K53 and K56 in bovine pancreatic PLA2 with the anionic interface. The maximum rate at saturating diheptanoylphosphatidylcholine micelles, VMapp, for WT, K56M, and K53M in 4 M NaCl is in the 800-1300 s-1 range. In contrast, VMapp at 0.1 M NaCl is considerably higher for K56M (400 s-1) and K53M (230 s-1) compared to the rate with WT (30 s-1) or K56E (45 s-1). The rate of hydrolysis of anionic dimyristoylphosphatidylmethanol vesicles is virtually the same with all these mutants (200-300 s-1) and it is not affected by added NaCl. The chemical step for the hydrolysis of anionic and zwitterionic substrates remains rate-limiting in the presence or absence of added NaCl. A modest (approximately 10-fold) effect of K56M substitution or of added NaCl is seen on the binding of the enzyme to the interface; however, the binding of the substrate or a substrate mimic to the active site of the enzyme at the interface is not affected by more than a factor of 2. Magnitudes of the primary rate and equilibrium parameters at the zwitterionic and anionic interfaces show that the effect of mutation or of added NaCl is primarily on k*cat at the zwitterionic interface. These results are interpreted in terms of a two-state model for the interfacial allosteric activation, where the enzyme-substrate complex at the zwitterionic interface becomes catalytically active only after the positive charge on cationic K56 and K53 has been removed by mutation or neutralized by anionic charges in the interface.