Clofarabine 5 -di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

摘要

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of a- and p-subunits that form active α_nβ_m (n, m = 2 or 6) complexes, a 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 (CIF), 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 CIF di- and triphosphates (CIFDP and CIFTP) are presented. CIFTP is a reversible inhibitor (K, = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by CIFTP, suggesting its A site binding. CIFDP is a slow-binding, reversible inhibitor (K_i～* = 17 nM; t_(1/2)= 23 min). CDP protects a from its inhi bition. The altered off-rate of CIFDP from E • CIFDP～* by CIFTP (A site) or dGTP (S site) and its inhibition of D57N-α together im plicate its C site binding. Size exclusion chromatography of hRNR or a alone with CIFDP or CIFTP, ±ATP or dGTP, reveals in each case that a forms a kinetically stable hexameric state. This is the first exam ple of hexamerization of a induced by an NDP analog that rever sibly binds at the active site.