Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Abstract

The X-ray structure of a mutant porcine pancreatic phospholipase A, inhibitor complex [Thunnissen et al. (1990) Nature 347, 689-6911 has been determined. This structure shows several interactions between the sn-2-acyl chain and the phosphate moiety of the inhibitor at sn-3 and the protein. The interactions of the remaining part of the polar head group are less clear. Because Arg53 is in close proximity to the head group, we tested the importance of charge at position 53 on enzymatic activity and specificity. Arg53 has been replaced by a glutamine and a glutamic acid in mutants R53Q and R53E, respectively. The effects of the mutations were tested with both zwitterionic and anionic substrates. With monomeric, zwitterionic, (R,S)-1,2-dihexanoyldithiopropyl-3-phosphocholine as substrate, the mutants R53Q and R53E display twofold and sevenfold, respectively, increased kJKm values, composed of increased k,, and decreased K , values. Tested on micelles of zwitterionic (R)-l,2-dioctanoylglycero-3-phosphocholine the mutants R53Q and R53E are more active than the native enzyme, whereas these mutations have an opposite effect on the activity on anionic (R)-l,2-dioctanoylglycero-3-phosphoglycol. Thus, whereas the native enzyme is 0.3 times as active on zwitterionic as on the anionic substrate, these ratios are 1.0 (R53Q) and 1.7 (R53E) for the mutants. No changes in activity were observed with the anionic substrate (R)-1,2-dioctanoylglycero-3-sulfate. Binding studies with substrate-derived inhibitors confirmed the increased affinity for zwitterionic phospholipids and the reduced affinity for anionic phospholipids. The kinetic and binding data indicate the involvement of the charge of residue 53 in head-group specificity and suggest a position of residue 53 closer to the choline or glycol than to the phosphate.The lipolytic enzyme phospholipase A, (PLA,) hydrolyzes the sn-2 ester bond of phosphoglycerides in a calcium-dependent and stereospecific reaction. Phospholipases occur both extracellularly and intracellularly. The extracellular enzymes are found abundantly in mammalian pancreas and in snake or bee venoms serving a digestive function. In addition the venom phospholipases show a wide range of pharmacological actions. The intracellular phospholipases are present in low concentrations in almost every mammalian cell and have an important role in inflammation processes by Abbreviations. PLA,, phospholipase A,; R53Q and R53E, mutants of PLA, with Arg53 changed to glutamine and glutamic acid, respectively ; Oco,GroSO,H, (R)-l,2-dioctanoylglycero-3-sulfate ; Oco,GroPGlo, (R)-l,2-dioctanoylglycero-3-phosphoglycol; Oc0,GroPCho, (R)-l,2-dioctanoylglycero-3-phosphocholine; Lau,GroPCho, (R)-l,2-didodecanoylglycero-3-phosphocholine; HxoS,GroPCho, (R,S')-1,2dihexanoyldithiopropyl-3-phosphocholine; DodPCho, n-dodecylphosphocholine; HxdPCho, n-hexadecylphosphocholine; MyrAholPCho, (R)-2-tetradecanoylaminohexanol-l-phosphocholine ; MyrAholPGlo, (R)-2-tetradecanoylaminohexanol-1 -phosphoglycol; KL, two-dimensional Michaelis Menten const...

Investigating porcine pancreatic phospholipase A2 action on vesicles and supported planar bilayers using a quartz crystal microbalance with dissipation

“…pPLA 2 contains positively charged residues involved in the interfacial binding [31,32], giving higher activity toward vesicles containing the anionic phospholipid PG than towards pure zwitterionic (PC) vesicles. Thus, in solution, the presence of PG stimulates the rate of breakdown of vesicles, as measured by dynamic light scattering (Fig.…”

Identification of Essential Residues for the Catalytic Function of 85-kDa Cytosolic Phospholipase A2

“…Arginine is known to interact with phosphate groups in several phosphohydrolases (e.g. alkaline phosphatase (27) and glycogen phosphorylase (28)) or the G-protein G i␣1 (29), and Arg-53 of pancreatic PLA 2 (40) plays an important role in catalysis, providing specificity in interaction with the phospholipid head group. By this model, we would expect that mutation of Arg-200 to lysine (the R200K mutant) would show, as it does, a significant drop in activity.…”

An Extended Binding Pocket Determines the Polar Head Group Specificity of Porcine Pancreatic Phospholipase A2

“…The X-ray study of the native bovine pancreatic PLA, showed the proximity of residue 53 and 56 to the choline head group (Tomoo et al, 1992). Site-directed mutagenesis showed that residues 53 (Lugtigheid et al, 1993b) and 56 (Noel et al, 1990(Noel et al, , 1991Lugtigheid et al, 1993 a) are important for head group binding and for the binding to interfaces. Site-directed mutagenesis showed that residues 53 (Lugtigheid et al, 1993b) and 56 (Noel et al, 1990(Noel et al, , 1991Lugtigheid et al, 1993 a) are important for head group binding and for the binding to interfaces.…”