Introduction of a C‐terminal aromatic sequence from snake venom phospholipases A₂ into the porcine pancreatic isozyme dramatically changes the interfacial kinetics

Abstract

Porcine pancreatic phospholipase A 2 (PLA 2 ) 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 PLA 2 s, which display high affinity for a zwitterionic interface. The PLA 2 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 app. max ) 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 app.max is most likely made up of a 24-fold reduction in catalytic turnover (k cat ) and 18-fold improved substrate binding (K m *).

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

“…1. PnLTA (250 g/ml) was incubated with PLA2 (50 g/ml) in a buffer (160 mM HEPES [pH 7.4] and 10 mM CaCl 2 ) for 24 or 48 h (17). Although the enzyme reaction with these PLA2 enzymes was inefficient compared to that with PAF-AH, the two PLA2 enzyme treatments produced new peaks with 264 to 267 mass units less than those of the original peaks (8,591 versus 8,324 m/z in Fig.…”

Neurotoxicity and Other Pharmacological Activities of the Snake Venom Phospholipase A2OS2: The N-Terminal Region Is More Important Than Enzymatic Activity

“…1) that would explain at least in part why the C-terminal chimera is less active than WT OS 2 (Table 3). Accordingly, mutation of asparagine to a tryptophan at this position (N117W) in the pancreatic group IB sPLA 2 results in a 18-fold increase in interfacial binding (78). OS 2 has a second tryptophan at position 75 ( Fig.…”

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

“…Mutants of human group IIA [41] and pancreatic group I PLA # s [42][43][44] have also shown the importance of tryptophan residues on the IBS for enhanced binding to both neutral and anionic vesicles. Of the AtxA Phe#% mutants studied in the present paper, F24W shows the highest enzymic activity, close to that of the wild type.…”