Luba J, Nare B, Liang P H, Anderson K S, Beverley S M, Hardy L W
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, Worcester 01605, USA.
Biochemistry. 1998 Mar 24;37(12):4093-104. doi: 10.1021/bi972693a.
Pteridine reductase 1 (PTR1) is a novel broad spectrum enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. 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 the pro-S hydride of NADPH to carbon 6 on the si face 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 upon V/K for 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.
蝶啶还原酶1（PTR1）是原生动物寄生虫利什曼原虫中一种新型的参与蝶呤和叶酸代谢的广谱酶。PTR1的过表达赋予这些原生动物对甲氨蝶呤的抗性，这源于该酶还原二氢叶酸的能力及其对甲氨蝶呤相对不敏感。催化反应的动力学机制和立体化学过程证实PTR1属于短链脱氢酶/还原酶（SDR）家族。以叶酸为底物时，PTR1催化两轮还原反应，生成5,6,7,8-四氢叶酸并氧化2当量的NADPH。在叶酸还原过程中二氢叶酸会短暂积累，它既是PTR1的底物也是抑制剂。PTR1将NADPH的前-S氢化物转移到二氢叶酸si面的C6位，产生与二氢叶酸还原酶相同立体异构体的四氢叶酸。产物抑制和同位素分配研究支持有序三元复合物机制，即NADPH首先结合，还原型蝶啶之后NADP +解离。其他SDR也有相同的动力学机制和NAD(P)H氢化物手性偏好。在高浓度二氢叶酸条件下，由转移氢化物的同位素取代引起的二氢叶酸还原反应中V/K的氚效应被抑制，这与稳态有序动力学机制一致。有趣的是，二元酶-NADPH复合物的一半似乎无法快速周转。荧光猝灭结果也表明存在无活性的二元酶-二氢叶酸复合物。PTR1与其底物之间观察到的无活性复合物在SDR家族成员中是独特的，可能为开发抗利什曼病疗法提供线索。
