6-tert-Butyl-2,3-epoxy-5-cyclohexene-1,4-dione (TBE), a metabolite of 3-tert-butyl-4-hydroxyanisole, was converted to 6-tert-butyl-2,3-epoxy-4(R)-hydroxy-5-cyclohexen-1-one ((4R)-TBEH) and 6-tert-butyl-2,3-epoxy-4(S)-hydroxy-5-cyclohexen-1-one ((4S)-TBEH) by TBE-reducing enzymes in rat liver cytosol. Two TBE-reducing enzymes (TBE-R1 and TBE-R2) were purified 18- and 117-fold, respectively, to apparent homogeneity from rat liver cytosol using DEAE-Sephacel, Blue Sepharose CL-6B, hydroxylapatite, and Sephadex G-100 column chromatography. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both enzymes were monomeric. The purified TBE-R1 and TBE-R2 had molecular weights of 37 and 35 kDa and isoelectric points of 6.5 and 5.8, respectively. Both enzymes had an optimum pH of about 5.5 with TEE as substrate. TBE-R1 utilized NADH or NADPH equally as cofactor, and the K-m values of NADH and NADPH for TEE with TBE-R1 were estimated to be 15 and 29 mu M, respectively. On the other hand, TBE-RB specifically utilized NADPH and the K-m value for TEE was estimated to be 92 mu M in the presence of NADPH. Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates. In addition, TBE-RS reduced and oxidized 3-ketosteroids at a higher rate in the presence of NAD(H) and/or NADP(H). Both enzyme activities were inhibited by quercitrin or p-chloromercuribenzoic acid, but little inhibition was observed with phenobarbital or pyrazole. Dicoumarol inhibited significantly TBE-R1 activity but not TBE-R2 activity. In the conversion of TEE to TBEH, TBE-R1 preferentially reduced TEE to (4R)-TBEH, whereas TBE-R2 preferred the reduction of TBE to(4S)-TBEH. (C) 1999 Academic Press.
