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

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Abstract
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 RDR 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.

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