Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs.
Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).
D000244:Adenosine Diphosphate / Q000378:metabolism; D002250:Carbon Radioisotopes; D003856:Deoxyuracil Nucleotides; D004229:Dithiothreitol / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology*; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D018139:Simplexvirus / Q000201:enzymology*; D013997:Time Factors; D014316:Tritium; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D002250:Carbon Radioisotopes; D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D014316:Tritium; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; C043945:2'-chloro-2'-deoxyuridine 5'-phosphate; D012264:Ribonucleotide Reductases; D004229:Dithiothreitol