Miles R W, Tyler P C, Furneaux R H, Bagdassarian C K, Schramm V L
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1998 Jun 16;37(24):8615-21. doi: 10.1021/bi980658d.
Genetic defects in human purine nucleoside phosphorylase cause T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. Compounds with features of the transition-state structure of purine nucleoside phosphorylase were synthesized and tested as inhibitors. The transition-state structure for purine nucleoside phosphorylase is characterized by (1) an elevated pKa at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and (2) oxocarbenium ion formation in the ribosyl ring (Kline, P. C., and Schramm, V. L. (1995) Biochemistry 34, 1153-1162). Both features have been incorporated into the stable transition-state analogues, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (immucillin-H) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1, 4-imino-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylase. The inhibitors exhibit equilibrium dissociation constants (Ki) from 23 to 72 pM and are the most powerful inhibitors reported for the enzyme. Complete inhibition of the homotrimeric enzyme occurs at one mole of inhibitor per mole of enzymic trimer. Binding of the transition-state inhibitor at one site per trimer prevents inhibitor binding at the remaining two sites of the homotrimer. A mechanism of sequential catalysis at each subunit, similar to that of F1 ATPase, is supported by these results. Slow inhibitor dissociation (e.g., t1/2 of 4.8 h) suggests that these compounds will have favorable pharmacologic properties. Interaction of transition-state inhibitors with purine nucleoside phosphorylase is different from reactant-state (substrate and product analogue) inhibitors of the enzyme which bind equally to all subunits of the homotrimer.
人类嘌呤核苷磷酸化酶的基因缺陷会导致以T细胞缺陷为主要表型的病症。有人提出，该酶的有效抑制剂可能会干预T细胞功能紊乱。合成了具有嘌呤核苷磷酸化酶过渡态结构特征的化合物，并将其作为抑制剂进行测试。嘌呤核苷磷酸化酶的过渡态结构的特征在于：（1）嘌呤环N7处的pKa升高，有利于质子化或与酶形成良好的氢键相互作用；（2）核糖环中形成氧碳鎓离子（克莱因，P.C.，和施拉姆，V.L.（1995年）《生物化学》34卷，1153 - 1162页）。这两个特征都已被纳入稳定的过渡态类似物中，即（1S）-1-（9-脱氮次黄嘌呤-9-基）-1,4-二脱氧-1,4-亚氨基-D-核糖醇（免疫菌素-H）和（1S）-1-（9-脱氮鸟嘌呤-9-基）-1,4-二脱氧-1,4-亚氨基-D-核糖醇（免疫菌素-G）。这两种抑制剂对小牛脾脏和人红细胞嘌呤核苷磷酸化酶均表现出缓慢起效的紧密结合抑制作用。这些抑制剂的平衡解离常数（Ki）在23至72皮摩尔之间，是该酶已报道的最强效抑制剂。每摩尔酶三聚体只需一摩尔抑制剂就能完全抑制该同三聚体酶。过渡态抑制剂在三聚体的每个位点结合会阻止其在同三聚体其余两个位点的结合。这些结果支持了每个亚基依次催化的机制，类似于F1 ATP合酶的机制。抑制剂缓慢解离（例如，半衰期为4.8小时）表明这些化合物将具有良好的药理特性。过渡态抑制剂与嘌呤核苷磷酸化酶的相互作用不同于该酶的反应物态（底物和产物类似物）抑制剂，后者与同三聚体的所有亚基均能平等结合。
