*2.3. Missense Mutants Impair CHS-Coupled ATPase Activity of ABCG5*/*G8*

Using the CHS-coupled ATPase activity as the functional readout, we initiated studies in the catalytic mechanism of ABCG5/G8 by exploiting the transporter's missense mutations that undergo proper trafficking to post-ER cell membranes (ER-escaped mutants). In this study, we used *Pichia pastoris* yeast and expressed recombinant proteins of G8-G216D, a catalytically deficient mutant [18], ABCG5E146Q/G8WT (G5-E146Q) and ABCG5WT/G8R543S (G8-R543S), two loss-of-function/sitosterolemia missense mutants [22,37], and ABCG5A540F/G8WT (G5-A540F), a loss-of-function mutant with putative sterol-binding defect [21] (Figure 1B and Figure S1).The purified mutants were preincubated with *E. coli* polar lipids and sodium cholate as described above. As shown in Figure 5, when compared with WT, the sitosterolemia missense mutants, G5-E146Q and G8-R543S, showed a ~80% reduction of the specific activity in CHS-coupled ATP hydrolysis (160 ± 15 nmol/min/mg and 150 ± 5 nmol/min/mg, respectively). The sterol-binding mutant G5-A540F, when compared to WT, showed a ~90% reduction of the specific activity in CHS-coupled ATP hydrolysis (90 ± 10 nmol/min/mg). Similar levels of activity reduction were also observed for non-CHS-coupled ATP hydrolysis (Figure S2). We then performed ATP concentration-dependent experiments and analyzed the Michaelis–Menten kinetics for these three mutants. For all mutants, KM(ATP) remained nearly the same as compared to WT, but the mutants displayed a 40–60% reduction in the catalytic rate (Table 1). This result suggests that the mutants do not alter their ability of the nucleotide association, and other molecular events contribute to the reduction of the specific ATPase activity.

The effects of CHS on ABCG5/G8 WT and mutants were further investigated by measuring the ATP hydrolysis in the CHS concentration-dependent manner at a saturated ATP concentration (5 mM here). Purified proteins were preincubated with *E. coli* polar lipids, sodium cholate, and a wide range of CHS concentrations (0.064 mM to 4.1 mM). For WT, we obtained a Vmax of 702.9 ± 50.7 nmol/min/mg, a KM(CHS) of 0.79 mM, and a kcat of 1.74 s−<sup>1</sup> (Figure 6 and Table 2). In the presence of *E. coli* polar lipids, the catalytic rates were similar between the CHS and ATP-dependent ATPase activities, with a Vmax of ~700 nmol/min/mg, which is about four times higher than that in the presence of liver polar lipids (Tables 1 and 2) and more than twofold higher than the previously reported value, ~290 nmol/min/mg [33]. The catalytic rates of the mutants decreased by 70–90%, except for G5-A540F, whereas both G5-E146Q and G8-R543S displayed significantly larger KM(CHS), up to a twofold increase. This suggests a more profound impact of sitosterolemia mutations on the ABCG5/G8 ATPase activity through sterol–protein interaction or structural changes.

**Figure 6.** CHS dependence of ABCG5/G8 ATPase activity. Purified proteins were assayed in the presence of *E. coli* polar lipids, and the specific activities of ATP hydrolysis were obtained by the CHS concentration-dependent experiments (0–4.1 mM CHS). The curves are fitted to the Michaelis–Menten equation (Prism 8), and, using two independently purified proteins, the means of at least two independent experiments along with standard deviations are plotted here. The kinetic parameters are listed in Table 2.

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**Table 2.** Dependence of ABCG5/G8 ATPase activity on cholesteryl hemisuccinate.

<sup>a</sup> Standard errors were calculated from the fits shown in Figure 6 using GraphPad Prism 8. <sup>b</sup> Turnover rates, kcat, were calculated using the following formula: Vmax = kcat × [E], where [E] is the protein concentration of ABCG5/G8 (363.1 nM). <sup>c</sup> Differential Gibbs free energy was calculated according to the following formula: ∆∆GMUT <sup>=</sup> <sup>−</sup>RTln(kMUT/kWT), where kMUT is the kcat of mutants, kWT is the kcat of WT, R <sup>=</sup> 8.314 J·mol−<sup>1</sup> ·K −1 (R: gas constant), and T = 310.15 K (37 ◦C). <sup>d</sup> Both WT and mutants were assayed in the presence of *E. coli* polar lipids. <sup>e</sup> Number of independent experiments.
