Structural Basis of the Anionic Interface Preference and Activation of Pancreatic Phospholipase A₂

Abstract

Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.

Crystal Structures of the Free and Anisic Acid Bound Triple Mutant of Phospholipase A2

“…In most of the bovine pancreatic PLA2 and its mutant structures studied so far, 5,22,27,33,34 the surface loop residues were always found to be disordered, except in the high-resolution (1.5 Å ) orthorhombic form 26 of the recombinant PLA2. Again in the present free triple mutant structure, the electron density is not clear and the surface loop residues are disordered.…”

“…13 -16 The importance and involvement of the interfacial binding residues in interfacial catalysis have been outlined. 17 -21 Yu et al 22 have suggested a concurrent role for several cationic residues, possibly Lys53, Lys56, Lys120 and Lys121, in determining the anionic interface preference of these diverse types of secreted PLA2. They have shown that the lysine-tomethionine substitution of residues 53, 56, 120 and 121 eliminates the anionic interface preference of the wild-type (WT) and enhances its preference for the zwitterionic interface.…”

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2

“…Numerous studies within the context of interfacial enzyme kinetics and activation have shown such native state heterogeneity for ppPLA 2 and the prominent role of its N-terminus therein [64][65][66][67]. The involvement of Trp3 in interfacial activation was confirmed by fluorescence measurements [68,69].…”

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin

“…The binding of PLA 2 to the surface of aggregated substrates increases substantially the enzyme activity, an effect known as interfacial activation (Gelb et al, 1995;Yu et al, 2000). Several lines of evidence show that PLA 2 activity and PLA 2 -lipid interaction are regulated by membrane-bound mode of PLA 2 and the physical properties of substrates, i.e., curvature, charge and fluidity (Tatulian, 2003;Winget et al, 2006;Pande et al, 2006;Ray et al, 2007;Chiou et al, 2010Chiou et al, , 2011a.…”