A model for the molecular mechanism of interfacial activation of phospholipase A₂ supporting the substrate theory

Abstract

Changes occurring in the activity of porcine pancreatic phospholipase A 2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2-di [6-(pyren-l-yl)butanoyl]-sn-glycero-3-phosphocholine or 1-[6-(pyren-1-yl)butanoyl]-2-[6-(pyren-1-yl)hexanoyl]-sn-glycero-3-phosphochofine were studied. A 2-fold enhancement was observed in the activity of phospholipase A~ towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A 2 activity at the critical mieellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A 2.

O‐ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum‐compatible DNA transfection agent

“…35 The hydrolysis of 0.6 µM EDPYPC in 0.1 M NaCl, 5 mM CaCl2, 5 mM Tris (pH 7) buffer containing 10 mM cholate by phospholipase A2 from Apis mellifera bee venom (Sigma) was measured with a Farrand spectrofluorometer with 5-nm slit widths. Various amounts of enzyme, up to 160 units, were used.…”

Exploring the Conformational States and Rearrangements of Yarrowia lipolytica Lipase

“…The interfacial mechanism followed by lipases can vary significantly depending on their structure and origin. Some lipases have been described to involve conformational changes upon adsorption at the lipid interface (36) or substrate binding (37). Other lipases have also been described to follow a two-step mechanism initiated by the adsorption of the enzyme at the lipid interface, followed by the formation of the enzyme/substrate complex.…”

Orientation and Conformation of a Lipase at an Interface Studied by Molecular Dynamics Simulations

“…The presence of a structured lipid interface with subtle physical properties adds another complexity to the problem of explaining the response of the enzyme when in contact with the interface. Different mechanisms of interfacial activation, which can be classified in two extremes, have been proposed (Thuren, 1988;Derewenda et al, 1994;Peters et al, 1995). One is based on a substrate-mediated mechanism (Thuren, 1988), suggesting that changes in the physical properties of the lipid substrate such as hydration, fluidity, curvature, charge, and composition trigger the activation, whereas the other proposed mechanism involves conformational changes in the enzyme upon adsorption to the lipid interface (Derewenda et al, 1994).…”

“…Different mechanisms of interfacial activation, which can be classified in two extremes, have been proposed (Thuren, 1988;Derewenda et al, 1994;Peters et al, 1995). One is based on a substrate-mediated mechanism (Thuren, 1988), suggesting that changes in the physical properties of the lipid substrate such as hydration, fluidity, curvature, charge, and composition trigger the activation, whereas the other proposed mechanism involves conformational changes in the enzyme upon adsorption to the lipid interface (Derewenda et al, 1994). It should be noted that these schemes are not mutually exclusive (Peters et al, 1995), and refined kinetic models involving both of them have been suggested (Brzozowski et al, 1991;van Tilbeurgh et al, 1993;Verger et al, 1984;Ransac et al, 1990Ransac et al, , 1991.…”