title: Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2'-chloro-2'-deoxyuridine 5'-triphosphate: a 3'-2' hydrogen transfer during the formation of 3'-keto-2'-deoxyuridine 5'-triphosphate.
abstract: The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP) into a mixture of 2'-deoxyuridine triphosphate (dUTP) and the unstable product 3'-keto-2'-deoxyuridine triphosphate (3'-keto-dUTP). This ketone can be trapped by reduction with NaBH4, producing a 4:1 mixture of xylo-dUTP and dUTP. When [3'-3H]ClUTP is treated with enzyme in the presence of NaBH4, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the 3H in ClUTP. Degradation of these isomeric nucleosides has established the location of the 3H in 3'-keto-dUTP as predominantly 2'(S). The xylo-dU had 98.6% of its label at the 2'(S) position and 1.5% at 2'(R). The isolated dU had 89.6% of its label at 2'(S) and 1.4% at 2'(R), with the remaining 9% label inferred to be at the 3'-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1000 mixture of dUTP and 3'-keto-dUTP, where the 3'-hydrogen of ClUTP is retained at 3' during production of dUTP and is transferred to 2'(S) during production of 3'-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin (Ashley et al., 1986) are discussed in terms of reductase being a model for the B12-dependent rearrangement reactions.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
authors.forename: G W
authors.initials: GW
authors.lastname: Ashley
authors.forename: G
authors.initials: G
authors.lastname: Harris
authors.forename: J A
authors.initials: JA
authors.lastname: Stubbe
mesh_terms: D003856:Deoxyuracil Nucleotides / Q000096:biosynthesis / Q000494:pharmacology; D006859:Hydrogen; D007778:Lactobacillus / Q000201:enzymology*; D008956:Models, Chemical; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; C058343:3'-keto-2'-deoxyuridine 5'-triphosphate; C037205:2'-chloro-2'-deoxyuridine 5'-triphosphate; D006859:Hydrogen; D012264:Ribonucleotide Reductases; C057295:ribonucleoside-triphosphate reductase