Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of ES, EP, and EI (KS, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.