CATALYTIC SIGNIFICANCE OF THE SPECIFICITY OF DIVALENT-CATIONS AS K-S-ASTERISK AND K(CAT)ASTERISK COFACTORS FOR SECRETED PHOSPHOLIPASE A(2)

Calcium is required for the substrate binding and for the chemical ste p of the interfacial catalytic turnover cycle of pancreatic phospholip ase A(2) (PLA2), but not for the binding of the enzyme to the interfac e. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and k(cat) for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyris toylphosphatidylmethanol (DMPM) vesicles is characterized as an equili brium dissociation constant for the interfacial binary (EC) and terna ry (ECL) complexes of PLA2 and substrate mimics (L). Of the cations(I I) that promote the binding of a mimic to the enzyme at the interface (E), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary ECL complexes with k(cat)* of <1 s(-1), compared to the rate of >100 s(-1) with Ca, Fe, Mn, Co, and Ni. Oxygen exchang e from (H2O)-O-18 to the products of hydrolysis of DMPM incorporates o ne O-18 in myristate. Incorporation of the first and second O-18 occur s during the incubation of both the products of hydrolysis in (H2O)-O- 18 with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of EC and E CL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn . These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that t he higher ground state stability of the ECS complex with the inhibito ry cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester car bonyl oxygen facilitates the near-attack geometry in the ECaS, and th e His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establ ishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.