Contributions of residues of pancreatic phospholipase A(2) to interfacial binding, catalysis, and activation

Primary rate and equilibrium parameters for 60 site-directed mutants of bov ine pancreatic phospholipase A(2) (PLA2) are analyzed so incremental contri butions of the substitution of specific residues can be evaluated. The magn itude of the change is evaluated so a functional role in the context of the N- and C-domains of PLA2 can be assigned, and their relationship to the ca talytic residues and to the i-face that makes contact with the interface. T he effect of substitutions and interfacial charge is characterized by the e quilibrium dissociation constant for dissociation of the bound enzyme from the interface (K-d), the dissociation constant for dissociation of a substr ate mimic from the active site of the bound enzyme (K-L*), and the interfac ial Michaelis constants, K-M* and k*(cat). Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decre ase in k(cat)* is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not p art of the catalytic site or the substrate binding pocket, also modulate k( cat)*. Interfacial anionic charge lowers K-d, and induces k(cat)* activatio n through K56, K53, K119, or K120. Significant changes in K-L* are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in K-M* [=(k(2)+k(-1))/k(1)] are attributed to k(cat)* (=k(2)) and K-L* (= k(-1)/k(1)). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on K-S*, and the effect of the interfacial anionic charge on k(cat)*. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivit y, and their allosteric regulation. We suggest that the interfacial recogni tion region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chem ical step.