Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit
Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit
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DOI：
10.1073/pnas.1013274108
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发表时间：
2011-06-14
影响因子：
11.1
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Aye, Yimon;Stubbe, JoAnne
人核糖核苷酸还原酶（hrnr）催化核苷酸转化为脱氧核苷酸，由α -和β -亚基组成，形成活性α (n) β (m) （n, m = 2或6）复合物。α结合NDP底物（CDP、UDP、ADP和GDP， C位点）以及控制酶活性（A位点）和底物特异性（S位点）的ATP和dntp （dATP、dGTP、TTP）变构效应物。氯法拉滨（ClF）是一种腺苷类似物，用于治疗难治性白血病。它的细胞毒性模式被认为与三磷酸作为hRNR的变构抑制剂的功能部分相关。本文对二磷酸和三磷酸ClF （ClFDP和ClFTP）抑制hRNR的机制进行了研究。ClFTP是一种可逆抑制剂（K-i = 40 nM），可快速灭活hRNR。然而，随着时间的推移，50%的活动被恢复。d57n - α是一个a位点改变的突变体，可以阻止ClFTP的抑制，表明其a位点结合。ClFDP是一种缓慢结合的可逆抑制剂(K*(i) = 17 nM；T(1/2) = 23分钟。CDP保护α免受其抑制。ClFTP （A位点）或dGTP （S位点）改变了ClFDP对E中心点ClFDP*的脱靶率，并抑制了d57n - α，这与ClFDP与C位点的结合有关。hRNR或α单独与ClFDP或ClFTP， +/- ATP或dGTP的尺寸排除色谱，显示在每种情况下α形成一个动力学稳定的六聚态。这是由NDP类似物诱导的α六聚化的第一个例子，该类似物在活性位点可逆结合。
Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of alpha- and beta-subunits that form active alpha(n)beta(m) (n, m = 2 or 6) complexes. alpha binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K-i = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-alpha, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (K*(i) = 17 nM; t(1/2) = 23 min). CDP protects alpha from its inhibition. The altered off-rate of ClFDP from E center dot ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-alpha together implicate its C site binding. Size exclusion chromatography of hRNR or alpha alone with ClFDP or ClFTP, +/- ATP or dGTP, reveals in each case that alpha forms a kinetically stable hexameric state. This is the first example of hexamerization of alpha induced by an NDP analog that reversibly binds at the active site.