Van Ness K P, McHugh T E, Bammler T K, Eaton D L
Department of Environmental Health, University of Washington, Seattle, Washington 98195, USA.
Toxicol Appl Pharmacol. 1998 Sep;152(1):166-74. doi: 10.1006/taap.1998.8493.
Mice constitutively express glutathione S-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.
小鼠在肝脏中组成性表达谷胱甘肽S-转移酶mGSTA3-3。这种同工型对黄曲霉毒素B1-8,9-环氧化物（AFBO）具有独特的高结合活性，从而保护小鼠免受黄曲霉毒素B1诱导的肝癌发生。相比之下，大鼠组成性表达一种密切相关的GST同工酶rGSTA3-3，其AFBO活性较低，因此对黄曲霉毒素B1暴露敏感。尽管这两种GST具有86%的序列同一性，并且对1-氯-2,4-二硝基苯（CDNB）具有相似的催化活性，但它们对AFBO的催化活性相差约1000倍。为了鉴定赋予对AFBO高活性的氨基酸，在被认为形成底物结合位点的两个区域中，将rGSTA3-3的非保守残基替换为mGSTA3-3的残基。使用九个不同残基的组合通过定点诱变产生了21种突变型rGSTA3-3酶。除了E208D突变体，rGSTA3-3的单突变产生的酶没有可检测到的AFBO活性。一般来说，随着mGSTA3-3残基被引入到rGSTA3-3酶中，AFBO结合活性以累加的方式增加，六个位点突变体E104I/H108Y/Y111H/L207F/E208D/V217K在所有突变酶中显示出最高的AFBO活性（40 nmol/mg/min）。当这种突变酶通过另外三个取代（D103E/I105M/V106I）进一步修饰时，AFBO结合活性降低了14倍，降至2.8 nmol/mg/min。尽管我们的rGSTA3-3突变体无法获得野生型mGSTA3-3的AFBO结合活性（265 nmol/mg/min），但我们能够鉴定出六个mGSTA3-3残基；Ile104、Tyr108、His111、Phe207、Asp208和Lys217，当它们共同取代rGSTA3-3时，对AFBO的谷胱甘肽结合活性显著增加（>200倍）。
