Leishmania major pteridine reductase 1 belongs to the short chain dehydrogenase family: stereochemical and kinetic evidence.
Leishmania major pteridine reductase 1 belongs to the short chain dehydrogenase family: stereochemical and kinetic evidence.
复制标题
利什曼原虫蝶啶还原酶 1 属于短链脱氢酶家族：立体化学和动力学证据。
DOI：
10.1021/bi972693a
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Hardy,LW
中科院分区：
文献类型：
--
作者：
Luba,J;Nare,B;Liang,PH;Anderson,KS;Beverley,SM;Hardy,LW
蝶啶还原酶1（Pteridine Reductase 1，PTR 1）是寄生于利什曼原虫体内的一种新的蝶呤和叶酸代谢酶。PTR 1的过表达赋予这些原生动物甲氨蝶呤抗性，这是由于该酶能够减少二氢叶酸及其对甲氨蝶呤的相对不敏感性。动力学机制和立体化学过程的催化反应确认PTR 1的短链脱氢酶/还原酶（SDR）家族的成员。以叶酸为底物，PTR 1催化两轮还原，产生5，6，7，8-四氢叶酸并氧化2当量的NADPH。二氢叶酸在叶酸还原过程中短暂积累，既是PTR 1的底物又是抑制剂。PTR 1将NADPH的前氢化物转移到二氢叶酸表面的碳6上，产生与二氢叶酸还原酶相同的THF立体异构体。产物抑制和同位素分配研究支持有序的三元复合物机制，NADPH结合第一和NADP+解离后，减少蝶啶。相同的动力学机制和NAD（P）H氢化物手性偏好与其他SDR。所观察到的氚效应uponV/K为减少所产生的同位素取代的转移氢化物的二氢叶酸被抑制在高浓度的二氢叶酸，符合稳态有序的动力学机制。有趣的是，一半的二元酶-NADPH复合物似乎不能快速周转。荧光猝灭结果也表明存在非生产性的二元酶-二氢叶酸复合物。在PTR 1和其底物之间观察到的非生产性复合物在SDR家族成员中是独特的，并可能为开发抗利什曼病治疗剂提供线索。
Pteridine reductase 1 (PTR1) is a novel broad spectrum enzyme of pterin and folate metabolism in the protozoan parasiteLeishmania. Overexpression of PTR1 confers methotrexate resistance to these protozoa, arising from the enzyme's ability to reduce dihydrofolate and its relative insensitivity to methotrexate. The kinetic mechanism and stereochemical course for the catalyzed reaction confirm PTR1's membership within the short chain dehydrogenase/reductase (SDR) family. With folate as a substrate, PTR1 catalyzes two rounds of reduction, yielding 5,6,7,8-tetrahydrofolate and oxidizing 2 equiv of NADPH. Dihydrofolate accumulates transiently during folate reduction and is both a substrate and an inhibitor of PTR1. PTR1 transfers thepro-Shydride of NADPH to carbon 6 on thesiface of dihydrofolate, producing the same stereoisomer of THF as does dihydrofolate reductase. Product inhibition and isotope partitioning studies support an ordered ternary complex mechanism, with NADPH binding first and NADP+dissociating after the reduced pteridine. Identical kinetic mechanisms and NAD(P)H hydride chirality preferences are seen with other SDRs. An observed tritium effect uponV/Kfor reduction of dihydrofolate arising from isotopic substitution of the transferred hydride was suppressed at a high concentration of dihydrofolate, consistent with a steady-state ordered kinetic mechanism. Interestingly, half of the binary enzyme−NADPH complex appears to be incapable of rapid turnover. Fluorescence quenching results also indicate the existence of a nonproductive binary enzyme−dihydrofolate complex. The nonproductive complexes observed between PTR1 and its substrates are unique among members of the SDR family and may provide leads for developing antileishmanial therapeutics.