Human plasma high-density lipoproteins (HDL), the primary vehicle for reverse cholesterol transport, are the target of serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes that turns serum opaque. HDL comprise a core of neutral lipidsscholesteryl esters and some triglyceridessurrounded by a surface monolayer of cholesterol, phospholipids, and specialized proteins [apolipoproteins (apos) A-I and A-II]. A HDL is an unstable particle residing in a kinetic trap from which it can escape via chaotropic, detergent, or thermal perturbation. Recombinant (r) SOF catalyzes the transfer of nearly all neutral lipids of ∼100,000 HDL particles (D ∼ 8.5 nm) into a single, large cholesteryl ester-rich microemulsion (CERM; D > 100 nm), leaving a new HDL-like particle [neo HDL (D ∼ 5.8 nm)] while releasing lipid-free (LF) apo A-I. CERM formation and apo A-I release have similar kinetics, suggesting parallel or rapid consecutive steps. By using complementary physicochemical methods, we have refined the mechanistic model for HDL opacification. According to size exclusion chromatography, a HDL containing nonlabile apo A-I resists rSOF-mediated opacification. On the basis of kinetic cryo-electron microscopy, rSOF (10 nM) catalyzes the conversion of HDL (4 μM) to neo HDL via a stepwise mechanism in which intermediate-sized particles are seen. Kinetic turbidimetry revealed opacification as a rising exponential reaction with a rate constant k of (4.400 ± 0.004) × 10 -2 min -1. Analysis of the kinetic data using transition state theory gave an enthalpy (Δ H‡), entropy (ΔS H‡), and free energy (ΔG H‡) of activation of 73.9 kJ/mol, -66.87 J/K, and 94.6 kJ/mol, respectively. The free energy of activation for opacification is nearly identical to that for the displacement of apo A-I from HDL by guanidine hydrochloride. We conclude that apo A-I lability is required for HDL opacification, LF apo A-I desorption is the rate-limiting step, and nearly all HDL particles contain at least one labile copy of apo A-I.
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