Stereoselective acetonyl side chain reduction of warfarin and analogs. Partial characterization of two cytosolic carbonyl reductases
Hermans, J.J.; Thijssen, H.H.
Drug Metabolism and Disposition the Biological Fate of Chemicals 20(2): 268-274
1992
ISSN/ISBN: 0090-9556 PMID: 1352220 Document Number: 390057
The reductase activity mediating the ketone reduction of the acetonyl side-chain of warfarin and analogs has been partially purified from rabbit liver cytosol. The reductase activity is resolved in two different fractions (A and B) by DEAE-Sephacel chromatography. Both fractions reduce the acetonyl group of warfarin analogs with marked substrate (R-enantiomers), as well as product stereoselectivity (alcohols of the S-configuration). The reductases are NADPH-dependent, which is absolute for fraction B. The enzyme kinetics of the R-enantiomers of warfarin and three 4'-derivatives (4'-nitro, 4'-chloro-, and 4'-methoxywarfarin) has been investigated. In contrast to fraction B, fraction A is sensitive in its KM for 4' substitution: the KM values of 4'-nitro- and chloro-analogs are .apprx.6 times lower than the KM values of the 4'-methoxy analog or warfarin itself. On the other hand, the Vmax values of fraction A are all in the range of about 1 to 2 nmol/mg .times. min, whereas the Vmax values of fraction B vary from about 1 (4'-methoxywarfarin) to 12 (the 4'-nitro analog). The intrinsic activities (Vmax/KM) of both enzymes show the same rank order: 4'-nitro > 4'-chloro > 4'-methoxy = warfarin. Warfarin reductase activity of both enzymes is not inhibited by pyrazole, sodium barbitone, or dicoumarol, but is strongly inhibited by quercetin, indomethacin, furosemide, and prostaglandin E2 (PGE2). In addition, fraction A is inhibited by menadione and androsterone, fraction B is inhibited by estrone. Various compounds were tested as substrates for these enzyme fractions. Menadione, haloperidol, glyceraldehyde, and estrone are substrates for fraction A but not for fraction B. In contrast, androsterone is a (weak) substrate for fraction B only. p-Nitrobenzaldehyde, p-nitroacetophenone, and PGE2 are reduced by both enzymes. The KM values for the latter substrate agree well with the Ki values that PGE2 has a (competitive) inhibitor of R-4-nitrowarfarin reduction by both enzyme fractions. The product from PGE2 reduction by both enzyme fractions is prostaglandin F2.alpha. The highly stereoselective acetonyl side-chain reduction of warfarin analogs by rabbit liver cytosol is conducted by at least two enzymes. Endogenous substrates for both enzymes may be prostaglandins and steroids.