Khojasteh-Bakht S C, Nelson S D, Atkins W M
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, 98195-7610, USA.
Arch Biochem Biophys. 1999 Oct 1;370(1):59-65. doi: 10.1006/abbi.1999.1361.
(R)-(+)-Menthofuran is the proximate toxic metabolite of pulegone, the major constituent of the pennyroyal oil, that contributes significantly to the hepatotoxicity resulting from ingestion of this folklore abortifacient pennyroyal oil. Recently, menthofuran was shown to be metabolized by cytochrome P450 to form (R)-2-hydroxymenthofuran. In this paper it is demonstrated that glutathione S-transferase (GST) catalyzes the tautomerization of 2-hydroxymenthofuran to mintlactone and isomintlactone, apparently without the formation of stable glutathione (GSH) conjugates. The reaction strictly required GSH; S-methyl GSH, which binds to the active site and leaves the active site Tyr-9 partly ionized, did not support GST-catalyzed isomerization. It was also determined that the tautomerization reaction requires the active site tyrosine, Tyr-9. The rat GSTA1-1 mutant (Y9F), with the active site tyrosine replaced with phenylalanine, demonstrated no catalytic activity. Rat cytosolic GST A1-1, in the presence of GSH, tautomerized 2-hydroxymenthofuran with apparent K(M) and V(max) values of 110 microM and 190 nmol/min/nmol GST, respectively. However, the site-directed mutant (F220Y), in which Tyr-9 and GSH in the binary complex [GST. GSH] have lower pK(a)s, exhibited K(M) and V(max) values of 97 microM and 280 nmol/min/nmol GST, respectively. Similarly, human liver cytosol catalyzed the tautomerization of 2-hydroxymenthofuran in a GST-dependent reaction. The mechanism most consistent with the data is a general-base catalyzed isomerization with GS(-) serving to deprotonate the substrate to initiate the reaction.
(R)-(+)-薄荷呋喃是长叶薄荷酮的直接毒性代谢产物，长叶薄荷酮是薄荷油的主要成分，大量摄入这种民间堕胎药薄荷油会导致肝毒性，薄荷呋喃在其中起了重要作用。最近研究表明，细胞色素P450可使薄荷呋喃代谢生成(R)-2-羟基薄荷呋喃。本文证明，谷胱甘肽S-转移酶（GST）催化2-羟基薄荷呋喃互变异构形成薄荷内酯和异薄荷内酯，显然没有形成稳定的谷胱甘肽（GSH）共轭物。该反应严格需要GSH；S-甲基谷胱甘肽结合到活性位点并使活性位点的酪氨酸-9部分离子化，不能支持GST催化的异构化反应。还确定互变异构反应需要活性位点的酪氨酸-9。大鼠GSTA1-1突变体（Y9F），其活性位点的酪氨酸被苯丙氨酸取代，没有催化活性。在GSH存在下，大鼠胞质GST A1-1使2-羟基薄荷呋喃互变异构，其表观K(M)和V(max)值分别为110 microM和190 nmol/min/nmol GST。然而，定点突变体（F220Y），其二元复合物[GST·GSH]中的酪氨酸-9和GSH的pK(a)较低，其K(M)和V(max)值分别为97 microM和280 nmol/min/nmol GST。同样，人肝细胞溶胶在GST依赖的反应中催化2-羟基薄荷呋喃的互变异构。与数据最一致的机制是一般碱催化的异构化反应，GS(-)使底物去质子化以启动反应。
