title: Clofarabine 5'-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit.
abstract: Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active α(n)β(m) (n, m = 2 or 6) complexes. α 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-α, 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)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Yimon
authors.initials: Y
authors.lastname: Aye
authors.forename: Joanne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000494:Allosteric Regulation; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D001087:Arabinonucleosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D000077866:Clofarabine; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D011756:Diphosphates; D011122:Polyphosphates; D021122:Protein Subunits; D000077866:Clofarabine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C005692:triphosphoric acid