Busulfan-Glutathione Conjugation Catalyzed by Human Liver Cytosolic Glutathione S-Transferases¹

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-α1-1. Human liver microsomes and cytosol were incubated with 40 µm busulfan and 1 mm GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT⁺) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT⁺ formation increased with pH (pH range, 6.50–7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K_m for busulfan was not determined. The intrinsic clearance (V_max/K_m) of busulfan conjugation was 0.167 µl/min/mg with 50–1200 µm busulfan and 1 mm GSH. GSH V_max and K_m for busulfan conjugation were 30.6 pmol/min/mg and 312 µm, respectively. Ethacrynic acid (0.03–15 µm) inhibited cytosolic busulfan-conjugating activity with 40 µm busulfan and 1 mm GSH. Enzyme-mediated THT⁺ formation was decreased 97% by 15 µm ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-α1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 µl/min/mg, respectively. Busulfan is a GST substrate with a high K_m relative to concentrations achieved clinically (1–8 µm).