High Affinity, Stability, and Lactonase Activity of Serum Paraoxonase PON1 Anchored on HDL with ApoA-I ² Leonid Gaidukov and Dan S. Tawfik* Department of Biological Chemistry, Weizmann Institute of Science, RehoVot 76100, Israel ReceiVed May 10, 2005; ReVised Manuscript ReceiVed June 15, 2005 ABSTRACT: Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K d , 10 -9 M) and slow dissociation rates (t 1/2 > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K d ) 1.2 × 10 -7 M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by e20-fold to k cat /K M values of 10 6 -10 7 M -1 s -1 , while apoA-IV and apoA-II have almost no effect. Overall, the results indicate the high stability, selectivity, and catalytic proficiency of PON1 when anchored onto apoA-I HDL, toward lactone substrates, and lipophilic lactones in particular. Serum paraoxonase (PON1) 1 belongs to a family of enzymes that catalyze the hydrolysis of a broad range of carboxy esters, carbonates, and lactones, as well as toxic organophosphates, including the insecticide paraoxon (1, 2). An emerging body of evidence indicates that PON1 possesses important antiatherogenic roles. Human serum PON1 levels and catalytic proficiencies are inversely proportional to the risk of coronary heart disease (3, 4), and PON1 knockout mice are highly susceptible to atherosclerosis (5). PON1 is known to reside on the high-density lipoprotein (HDL) cholesterol-carrying particles, which play a key role in neutralizing potentially toxic, hydrophobic components of plasma, including oxidized lipids (6). The PON1-HDL complex is thought to protect against oxidative modifications, mediate the efflux of cholesterol from macrophages, and reduce the level of oxidized lipid forms involved in the development of atherosclerosis (7-9). The endogenous substrate(s) and mechanism of the anti- atherogenic activity of PON1 remain largely unknown. It is becoming apparent, however, that despite its traditional assignment as paraoxonase/arylesterase, PON1 is in fact a lactonase. PON1 has been shown to catalyze the hydrolysis of a variety of lactones (2, 10), as well as lactone formation (11). Moreover, laboratory evolution (12) and structure- reactivity studies of PON1 (13) indicated that PON1 is primarily a lactonase. In fact, the lactonase activity is the only activity common to all members of the PON family, and directed evolution of PON1 has led to PON1 variants with activity patterns and active site residues that resemble those of PON2 or PON3 (i.e., high lactonase/esterase activity and very low paraoxonase activity) (12, 14). Thus, lactones derived from fatty acid oxidation products may comprise the native substrates of PON1 (10, 15). PON1 is synthesized in the liver and secreted into the blood where it is associated with HDL-type complexes (16, 17). PON1’s localization on HDL seems to be critically important for its activity in vivo, probably by stabilizing the enzyme and providing an optimal environment for the interaction with its physiological substrates. PON1 can also associate with other amphiphilic complexes such as phos- pholipid and detergent micelles (18-20). PON1’s hydro- phobic N-terminus is compatible with a transmembrane helix (dubbed H1), and was suggested to mediate the anchoring of PON1 to HDL and phospholipid micelles (14, 18, 21). The crystal structure of a PON1 indicated an adjacent ² This work was supported by the Benoziyo Institute of Molecular Medicine. D.S.T. is the incumbent of the Elaine Blond Career Development Chair. * To whom correspondence should be addressed. Phone: +972 8 934 3637. Fax: +972 8 934 4118. E-mail: tawfik@weizmann.ac.il. 1 Abbreviations: PON1, serum paraoxonase; rePON1, recombinant PON1; HDL, high-density lipoprotein; rHDL, reconstituted HDL; Apo, apolipoprotein; PC, egg L-R-phosphatidylcholine; FC, free (unesterified) cholesterol; DPPE, dipalmitoylphosphatidylethanolamine; SPR, surface plasmon resonance; RU, resonance units; SUV, small unilamellar vesicles; 5-HETEL, 5(S)-hydroxy-6(E,Z),11(Z),14(Z)-eicosatetraenoic acid, 1,5-lactone; LCAT, lecithin cholesterol acyltransferase. 11843 Biochemistry 2005, 44, 11843-11854 10.1021/bi050862i CCC: $30.25 © 2005 American Chemical Society Published on Web 08/16/2005