Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site-directed mutations at sites thought to be involved in interfacial binding. Charged and polar residues in the C-terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface. The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild-type enzyme. Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (V(max)/(app.)) with the zwitterionic substrate were between 25 and 75% of that of the wild-type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (K(m)(*)) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in V(max)/(app.) is most likely made up of a 24-fold reduction in catalytic turnover (k(cat)) and 18-fold improved substrate binding (K(m)(*)).
CITATION STYLE
Janssen, M. J. W., Burghout, P. J., Verheij, H. M., Slotboom, A. J., & Egmond, M. R. (1999). Introduction of a C-terminal aromatic sequence from snake venom Phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics. European Journal of Biochemistry, 263(3), 782–788. https://doi.org/10.1046/j.1432-1327.1999.00557.x
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