Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift.
Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.
D001894:Borohydrides / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D003856:Deoxyuracil Nucleotides / Q000032:analysis / Q000138:chemical synthesis* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D014316:Tritium
D001894:Borohydrides; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D014316:Tritium; C048032:2'-chloro-2'-deoxyuridine 5'-diphosphate; C048033:2'-deoxy-3'-ketouridine 5'-diphosphate; D012264:Ribonucleotide Reductases