Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate

Abstract

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, and catalyzes the conversion of nucleotides to deoxynucleotides. The mechanism of inactivation of RDPR by 2'-azido-2'-deoxynucleoside 5'-diphosphate (N3UDP) has been examined using a variety of isotopically labeled derivatives: (1'-, 2'-, 3'-, or 4'-[2H])-N3UDPs and 2'-[15N3, 13C]-N3UDP. Electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopy studies using these compounds indicate that the 2' carbon-nitrogen bond to the azide moiety is cleaved prior to or upon formation of the nitrogen-centered radical derived from the azide moiety of N3UDP. EPR studies reveal no hyperfine interactions of the nitrogen-centered radical with the 1', 2', 3', or 4' hydrogens of N3UDP. ESEEM studies however, reveal that the 1' and 4' deuterons are 3.3 +/- 0.2 and 2.6 +/- 0.3 A, respectively, from the nitrogen-centered radical. Further support for carbon-nitrogen bond cleavage is derived from studies of the interaction of oxidized R1, C225SR1, and C462SR1 with R2 and N3UDP. In all three cases, in contrast to the results with the wild type R1, azide is detected. Nitrogen-centered radical is not observed with either oxidized R1 or C225SR1 but is observed with C462SR1. These results suggest that C225 is required for the conversion of azide into N2 and a nitrogen-centered radical. The dynamics of the inactivation of RDPR by N3UDP have also been examined. Use of [3'-2H]N3UDP reveals an isotope effect of approximately 2 on the loss of the tyrosyl radical and the rate of inactivation of RDPR. In both cases the kinetics are complex, suggesting multiple modes of inactivation. In addition, several modes of inactivation are required to explain the observation that loss of the tyrosyl radical is slower than the rate of inactivation. Studies using [5'-3H]N3UDP reveal that the rapid inactivation is the result of the formation of a tight noncovalent complex between modified nucleotide, nitrogen-centered radical and RDPR. Destruction of the nitrogen-centered radical is a slow process which appears to be accompanied by decomposition of the modified nucleotide into PPi, uracil, and 2-methylene-3(2H)-furanone. The latter covalently modifies R1 and ultimately leads to loss of approximately 50% of the activity of R1.

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…2 D-F). In retrospect, we have previously seen this type of behavior with another substoichiometric mechanism based inhibitor: 2Ј-azido-2Ј-deoxynucleotides, N 3 NDP (19,37,38). During a 2-min incubation of RNR with [5Ј-3 H] N 3 NDPs, Ͼ90% of RNR activity is lost, 50% of Y • is lost, and 0.7 eq radiolabel are associated with RNR.…”

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

“…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. The N-centered radical generated in this latter step has only recently been characterized through a combination of high-field EPR spectroscopy and DFT calculations (Fritscher et al, 2005).…”

Biomimetic Modeling of the Nitrogen-centered Radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides.

“…On the basis of the results, it was proposed the thiyl radical at C225 13 reacted with the released hydrazoic acid (HN 3 ) to form a sulfinylimine radical 14. 24 In summary, the experimental data suggested that the inhibitory mechanism of RNR by N 3 UDP started with the abstraction of a hydrogen atom from the carbon at C3' by cysteinyl radical C439 to give 11. However, rather than releasing H 2 O upon oxidizing the hydroxyl group on C3', azide ion was released to form the ketyl radical 12.…”

“…By double labeling of N 3 NDPs with 13 C and 15 N 3 and incubating with RDPR, Stubbe and Robins reported in 1993 that there was no hyperfine interaction between the nitrogen-centered radical and the 13 C nucleus, which suggested the cleavage of the nitrogen bond on the 2'-carbon (11⇒12). 24 Incubation of oxidized R1 and mutated cysteine proteins C225SR1 and C462SR1 with N 3 UDPs suggested the release of N 3 -, N 3 • , or HN 3 . On the basis of the results, it was proposed the thiyl radical at C225 13 reacted with the released hydrazoic acid (HN 3 ) to form a sulfinylimine radical 14.…”

“…1,18 The last step of the mechanism is the regenerating of thiyl radical on cys439 by the radical on C3' abstracting the hydrogen from cys439. 18,24 (Figure 3) Deoxynucleotides then leave the active site allowing for other ribonucleotides to enter and to be reduced by RNRs. The cys225-cys462 disulfide unit formed during the reduction is reduced to regenerate the free sulfhydryl groups by the two cysteine residues located at the end of the N-terminal of the R1 protein, cys754 and cys759, in order to continue the mechanistic cycle.…”