Mechanism of inactivation of Escherichia coli and Lactobacillus leichmannii ribonucleotide reductases by 2'-chloro-2'-deoxynucleotides: evidence for generation of 2-methylene-3(2H)-furanone.
Incubation of 2'-chloro-2'-deoxy[3'-3H]uridine 5'-diphosphate ([3'-3H]ClUDP) with Escherichia coli ribonucleotide reductase (RDPR) and use of thioredoxin-thioredoxin reductase as reductants result in release of 4.7 equiv of 3H2O/equiv of B1 protomer, concomitant with enzyme inactivation. Inactivation is accompanied by the production of 6 equiv of inorganic pyrophosphate [Stubbe, J. A., & Kozarich, J.W. (1980) J. Am. Chem. Soc. 102, 2505-2507] and by the release of uracil as previously shown [Thelander, L., Larsson, A., Hobbs, J., & Eckstein, F. (1976) J. Biol. Chem. 251, 1398-1405]. Reisolation of RDPR by Sephadex chromatography and analysis by scintillation counting indicate that 0.96 equiv of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. Incubation of [5'-3H]ClUDP with RDPR followed by similar analysis indicates that 4.6 mol of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. No 3H2O is released, and 6 equiv of inorganic pyrophosphate is produced during the inactivation. RDPR is protected against inactivation when dithiothreitol (DTT) is used as a reductant in place of thioredoxin-thioredoxin reductase. Incubation of [5'-3H]ClUDP with RDPR and DTT results in the isolation of CHCl3-extractable material that exhibits infrared absorptions at 1710 and 1762 cm-1. The infrared spectrum and the NMR spectrum of the CHCl3-extracted material are very similar to model compounds prepared by the interaction of 2-methylene-3(2H)-furanone with ethanethiol. Incubation of ribonucleoside-triphosphate reductase (RTPR) from Lactobacillus leichmannii with [3'-3H]ClUTP and 3 mM DTT also results in time-dependent 3H2O release concomitant with enzyme inactivation. Reisolation of the inactive protein by Sephadex chromatography followed by radiochemical analysis indicates that 0.4 equiv of 3H is bound covalently per mol of inactivated enzyme. Similar studies with [5'-3H]ClUTP indicate that 2.9 equiv of 3H is bound covalently per mol of inactivated enzyme. No 3H2O is released. High concentrations of DTT protect the enzyme against inactivation. Extraction of the enzymatic reaction mixture with CHCl3 and analysis of the isolated products result in an infrared spectrum and an NMR spectrum remarkably similar to those observed with the E. coli RDPR. Data presented are consistent with the proposal that both the E. coli and L. leichmannii enzymes are able to catalyze the breakdown of the appropriate 2'-chloro-2'-deoxynucleotide to a 3'-keto-2'-deoxynucleotide that can collapse to form the reactive sugar intermediate 2-methylene-3(2H)-furanone.(ABSTRACT TRUNCATED AT 400 WORDS)
D003856:Deoxyuracil Nucleotides; D004229:Dithiothreitol; D004926:Escherichia coli / Q000201:enzymology*; D005663:Furans / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D014500:Uracil Nucleotides / Q000494:pharmacology*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*
D003856:Deoxyuracil Nucleotides; D005663:Furans; D014500:Uracil Nucleotides; D014530:Uridine Diphosphate; C043946:2-methylene-3(2H)-furanone; C043945:2'-chloro-2'-deoxyuridine 5'-phosphate; D012264:Ribonucleotide Reductases; D004229:Dithiothreitol