Perspectives on Genetic Testing and Return of Results from the First Cohort of Presymptomatically Tested Individuals At Risk of Huntington Disease.

This qualitative study gathered opinions about genetic testing from people who received presymptomatic testing for Huntington's disease (HD) 20-30 years ago and have lived with the implications of that testing for decades. During the last section of a semi-structured interview, participants were asked open-ended questions about their opinions on the importance of autonomy in the decision to be tested for HD, whether a formal HD testing protocol is necessary, whether physician ordering for HD is acceptable without a formal protocol, whether online direct-to-consumer (DTC) genetic testing for HD is acceptable, and whether incidental/secondary findings should be returned in the context of whole exome/genome sequencing. Most-but not all-participants were in favor of an individual's right to decide whether and when to pursue HD testing, use of a formal HD testing protocol, and returning medically actionable secondary findings. However, the majority of participants were opposed not only to physician ordering and DTC HD testing in the absence of a formal protocol but also to returning a secondary finding of an expanded HD allele. This study presents the opinions of a unique and extremely well-informed cohort on issues that need to be taken into careful consideration by genetic counselors and other medical professionals who are developing genetic testing protocols, making decisions about the availability of genetic tests, and making decisions about whether and how to return incidental findings.

Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

O2 activation by non-heme diiron proteins: identification of a symmetric mu-1,2-peroxide in a mutant of ribonucleotide reductase.

Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O-O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 --> Glu and the surface mutation Trp48 --> Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe-O2-Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Delta9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664-14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.

Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2.

Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormö, M., Nordlund, P., & Sjöberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

Evidence for a glutathionyl-enzyme intermediate in the amidase activity of the bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli.

Glutathionylspermidine (Gsp) is a metabolite common to Escherichia coli and protozoal parasites of the Trypanosoma family. Though its role in E. coli is unknown, Gsp is known to be an intermediate in the biosynthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), a metabolite unique to trypanosomatids that may allow the parasites to overcome oxidative stresses induced by host defense mechanisms. The bifunctional Gsp-synthetase/amidase from E. coli catalyzes both amide bond formation and breakdown between the N1-amine of spermidine [N-(3-aminopropyl)-1,4-diaminobutane] and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly), with net hydrolysis of ATP [Bollinger et al. (1995) J. Biol. Chem. 270 (23), 14031-14041]. Synthetase and amidase activities reside in separate domains of the protein, and liberation of the amidase domain from the synthetase domain activates the amidase activity as much as 70-fold in kcat/K(m) for a chromogenic substrate gamma-Glu-Ala-Gly-pNA [Kwon et al., (1997) J. Biol. Chem. 272 (4), 2429-2436]. When substrates for the Gsp-synthetase activity are present (GSH, ATP-Mg2+), Gsp-amidase is highly activated (15-fold). We provide kinetic and mutagenesis evidence suggesting that the amidase operates by a nucleophilic attack mechanism involving cysteine as the catalytic nucleophile. Stopped-flow studies on the 25 kDa Gsp-amidase fragment and the 70 kDa full-length Gsp-synthetase/amidase with gamma-Glu-Ala-Gly-ONp demonstrate burst kinetics characteristic of a covalent acyl-enzyme intermediate. Studies using various group-specific protease inhibitors, such as iodoacetamide, suggest an active-site cysteine or histidine as being relevant to amidase activity, and site-directed mutagenesis indicates that Cys-59 is essential for amidase activity.

Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains.

The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1, 4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the opposing hydrolysis of this amide bond (Bollinger, J. M., Jr., Kwon, D. S., Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) J. Biol. Chem. 270, 14031-14041). In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains. In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules. A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein. A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs. This observation suggests that the amidase activity in the full-length protein is negatively autoregulated. The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage. No metal ion is required for the amidase activity. A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki approximately 10 microM) but does not inhibit the amidase activity.

Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of the metal centers in transcription.

SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli sigma 70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.

Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase.

Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W. (1975) J. Biol. Chem. 250, 2648-2654). Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.

Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase.

The SF-Abs, RFQ-EPR, and RFQ-Möss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary. Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent. A number of problems can be associated with the RFQ approach. For example, isopentane could conceivably interfere with or alter the chemistry to be studied. A second potential problem involves temperature-dependent equilibria among different intermediate species. This problem has been encountered by Dooley et al. with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers. This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general. Each of the three methods employed can yield time-resolved quantitation of reaction components. In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis. Obvious advantages to the RFQ-Möss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron. In our hands, quantitation by RFQ-EPR was most problematic and least reproducible. This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density. As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator. Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands. Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem. Time-resolved Möss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available. All of the iron centers can be examined simultaneously. The problems associated with Möss arise from its extreme insensitivity. It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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.

Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.

Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase.

Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.

2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase.

It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

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

The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type.