High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I

Leonid Gaidukov, Dan S Tawfik
Biochemistry 2005 September 6, 44 (35): 11843-54
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 x 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 <or=20-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.

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