Structural basis of substrate recognition in thiopurine s-methyltransferase.
Structural basis of substrate recognition in thiopurine s-methyltransferase.
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DOI：
10.1021/bi800102x
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发表时间：
2008-06
期刊：
影响因子：
2.9
通讯作者：
Yi Peng;Q. Feng;D. Wilk;A. Adjei;O. Salavaggione;R. Weinshilboum;V. Yee
中科院分区：
文献类型：
--
作者：
Yi Peng;Q. Feng;D. Wilk;A. Adjei;O. Salavaggione;R. Weinshilboum;V. Yee
硫代嘌呤S甲基转移酶(TPMT)以S-腺苷-L-蛋氨酸为供体，通过甲基化来调节6-巯基嘌呤等硫代嘌呤前药的细胞毒效应。具有TPMT变异等位酶的患者表现出蛋白质和/或酶活性水平降低，并面临硫嘌呤药物诱导毒性的风险。我们测定了小鼠TPMT的两种晶体结构，一种是产物S-腺苷-L-同型半胱氨酸的二元络合物，另一种是S-腺苷-L-同型半胱氨酸和底物6-巯基嘌呤的三元络合物，分辨率分别为1.8A和2.0A。结构的比较表明，活性部位环在6-巯基嘌呤结合时变得有序。两个配体的位置与预期的S氮气反应机理一致。Arg147和Arg221是唯一接近6-巯基嘌呤的极性氨基酸，是底物去质子化的可能参与者。为了探索这些残基对催化是否重要，在人类酶中制备了点突变。用谷氨酸取代Arg152(小鼠TPMT中的Arg147)可降低6-巯基嘌呤的Vmax，增加Km，但不能使S-腺苷-L-蛋氨酸的Km增大。在该位置用丙氨酸或组氨酸替换以及类似的Arg226替换(小鼠TPMT中的Arg221)不会对酶活性产生影响。双突变株Arg152Ala/Arg226Ala对6-巯基嘌呤的Vmax降低，Km增大。这些观察表明，Arg152或Arg226可能以某种方式参与TPMT反应，一个残基在另一个残基改变时补偿，Arg152可能比Arg226更直接地与底物相互作用，这与在小鼠TPMT晶体结构中的观察一致。
Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl- l-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl- l-homocysteine and as a ternary complex with S-adenosyl- l-homocysteine and the substrate 6-mercaptopurine, to 1.8 and 2.0 A resolution, respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S N2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases V max and increases K m for 6-mercaptopurine but not K m for S-adenosyl- l-methionine. Substitution at this position with alanine or histidine and similar substitutions of Arg226 (Arg221 in murine TPMT) result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased V max and increased K m for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure.