The basis for k*(cat) impairment in prophospholipase A(2) from the anion-assisted dimer structure

Kinetic results in this paper show that, contrary to earlier reports, pig p ancreatic prophospholipase A(2) (proPLA2) does not hydrolyze monodisperse s hort chain phosphatidylcholine below the critical micelle concentration. Pr oPLA2 is active on an anionic interface, but at a rate that is decreased by more than 100-fold compared to that of PLA2, the active form. Solution stu dies show that both proPLA2 and PLA2 bind to an anionic interface and also bind a tetrahedral intermediate mimic at the active site. The 1.5 Angstrom resolution crystal structure of the anion-assisted dimer of proPLA2 reporte d in this paper is compared with the corresponding structure for PLA2 [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. As a mimic for the forms b ound to the anionic interface, these structures provide insights into the p ossible structural basis for the impaired chemical step of the zymogen. The proPLA2 dimer contained within one crystallographic asymmetric unit has on e molecule of the inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-pho sphomethanol and is bridged by four coplanar sulfate anions. Relative to th e structure of PLA2, the subunit contact surface in proPLA2 displays a tilt ed orientation, an altered mode of inhibitor binding, displacement of a mec hanistically significant loop that includes Tyr69, and a critical active si te water seen in PLA2 that is not seen in proPLA2. These differences are in terpreted to suggest possible origins of the functional differences between the pro and active enzyme at an anionic interface. A structural origin of this difference is discussed in terms of the calcium-coordinated activated water mechanism of the esterolysis reaction. Together, a comparison of the structures of the anion-assisted dimers of PLA2 and proPLA2 not only offers an explanation of why the zymogen form is k*(cat)-impaired and binds poorl y even to the anionic interface but also supports a mechanism for the activ ated enzyme that includes a critical second-sphere assisting water bridging His48 and the calcium-coordinated catalytic water.