posted on 1998-08-15, 00:00authored byBao-Zhu Yu, Joseph Rogers, Gordon R. Nicol, Klaus H. Theopold, K. Seshadri, S. Vishweshwara, Mahendra Kumar Jain
Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic
turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the
interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate
binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of
dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant
for the interfacial binary (E*C) and ternary (E*CL) complexes of PLA2 and substrate mimics (L). Of
the cations(II) 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 E*CL complexes with kcat* of <1
s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the
products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second
18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not
with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of
E*C 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 the higher ground
state stability of the E*CS complex with the inhibitory cations increases the effective activation energy.
For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen
facilitates the near-attack geometry in the E*CaS, and the His-48·Asp-99 pair acts as a proton acceptor.
As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding
transition state are also discussed.