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

Abstract

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)

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

“…Similar UV-visible spectra were observed previously in incubations of wild type enzyme with 2Ј-substituted substrate analogues, and of suicidal C225S R1 protein in presence of R2 and substrate (11,18). The substrate-derived chro- mophore relates to R1 adducts of a highly reactive 2-methylene-3(2H)furanone intermediate (52), and is diagnostic for reactions involving suicidal decay of substrate radical intermediates formed by 3Ј carbon-hydrogen bond cleavage.…”

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

“…For disulfide substituents in the C29 position, experimental and computational studies are suggestive of homolytic cleavage, even in the enzyme-catalyzed reaction (Covè s et al, 1996;Pereira et al, 2005). However, even for halides, the influence of a polar, hydrogenbonding environment may be substantial enough to force the heterolytic elimination mechanism favored in the enzyme-catalyzed transformation (Harris et al, 1984;Fernandes and Ramos, 2003). Extensive work on the reaction of N 3 UDP with wild-type enzyme (Salowe et al, 1993;van der Donk et al, 1995) can be rationalized by either the anionic release of azide (Pereira et al, 2003) or by initial homolytic generation of an azide radical and subsequent reduction to azide, in both cases followed by a sequence of N 2 elimination and addition to the C39 carbon atom.…”

Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.

“…This model predicts that four dCDPs could be produced under single-turnover conditions, and the observation of three dCDPs was rationalized as resulting from incomplete prereduction of the cysteines in B1. This model also predicts that, under single-turnover conditions, a change in Cys-754 or Cys-759 to serine would prevent dCDP production whereas a change in either Cys-225 or Cys-230 should allow production of two dCDPs (4).Studies utilizing the mechanism-based inhibitor 2'-chloro-2'-deoxyuridine 5'-diphosphate (CIUDP) also make predictions concerning the function of the redox-active thiols (9,10). If the reduction process is impaired, but the general acid catalysis function of one of the thiols is retained, then the normal substrate CDP will be converted into a mechanismbased inhibitor (Fig.…”

“…Studies utilizing the mechanism-based inhibitor 2'-chloro-2'-deoxyuridine 5'-diphosphate (CIUDP) also make predictions concerning the function of the redox-active thiols (9,10). If the reduction process is impaired, but the general acid catalysis function of one of the thiols is retained, then the normal substrate CDP will be converted into a mechanismbased inhibitor (Fig.…”