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 lipidscholesteryl esters and some triglyceridesurrounded 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 approximately 100,000 HDL particles (D approximately 8.5 nm) into a single, large cholesteryl ester-rich microemulsion (CERM; D > 100 nm), leaving a new HDL-like particle [neo HDL (D approximately 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 microM) 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) x 10(-2) min(-1). Analysis of the kinetic data using transition state theory gave an enthalpy (DeltaH()), entropy (DeltaS(++)), and free energy (DeltaG()) 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.