On the Mechanism of Ribonucleoside Diphosphate Reductase from Escherichia coli EVIDENCE FOR 3°C-H BOND CLEAVAGE*

The 3’-carbon”hydrogen bond of [3’-3H]uridine 5‘- diphosphate is cleaved during its conversion to 2’-de-oxyuridine 5”diphosphate catalyzed by Escherichia coli ribonucleoside diphosphate reductase. A selection against 3H of approximately 3.3 is observed on this reduction reaction. During the course of this reaction, a small but significant amount of 3H is released to the solvent. Ribonucleoside-diphosphate reductase (EC cata-lyzes the reduction of ribonucleoside 5”diphosphates to their corresponding 2‘-deoxyribonucleoside 5“diphosphates. The Escherichia coli reductase has been characterized extensively by Reichard and co-workers review, Ref. and has been shown to consist of two nonidentical subunits, Bi (Mr = 78,000) and B:! (M, = 39,000) (2). The BI subunit binds substrates and allosteric effectors and contains sulfhydryl groups which are oxidized upon substrate reduction The BP subunit two nonheme irons (Fe(II1)) and an unusual organic free radical localized on a tyrosine residue (3-5). the laboratory of ribonucleoside diphosphate reductase with 2“chloro-2”deoxyuridine 5”diphosphate 2’-deoxy-2’-fluorocytidine

* This research was supported by Grants BC-285 and IN31R-03 from the American Cancer Society and by Grant CA-16359 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The mechanism shown in Scheme 1 demonstrates how such a 3"ketone could be generated during inactivation of ribonucleoside diphosphate reductase with 2'-ClUDP.'. This hypothetical reaction (Scheme l), based on analogy with chemical model systems (9), involves enzyme-catalyzed cleavage of the 2'-carbon"chlorine bond via a radical cation mechanism (7). In Scheme 1, the tyrosyl radical of BP abstracts the 3'-H atom of 2"ClUDP to generate the 3'-nucleotide radical 11. This initial radical abstraction facilitates cleavage of the 2'-C-C1 bond to generate radical cation 111. Radical cation I11 can then collapse to form 3'-ketone I. This mechanism predicts that the 3°C-H bond is broken during inactivation of ribonucleoside diphosphate reductase by 2"ClUDP.
A similar type of mechanism, that is cleavage of the 3'-C-H bond, might also be involved in the normal reduction of UDP to dUDP catalyzed by ribonucleoside diphosphate reductase. One method to test this hypothesis is the synthesis of [3'-'H]UDP and analysis for a 'H isotope effect. If cleavage of the 3°C-H bond is rate-determining or partially ratedetermining when UDP is reduced to dUDP, ['HIUDP would be reduced more rapidly than the [3H]UDP. The differences in rates of reduction of the 'H uersus 'H compound would be reflected in the differences in specific activities of starting material and product. We wish to report that incubation of [3'-'H]UDP with ribonucleoside diphosphate reductase results in a selection against 'H of approximately 3.3 on conversion of substrate into product. This observation supports the hypothesis that 3'-H of the substrate is involved in catalysis.

MATERIALS AND METHODS
Ribonucleoside diphosphate reductase was isolated using a dATP-Sepharose affinity column (10). Thioredoxin and thioredoxin reductase were isolated by modifications of procedures previously described Bradford (14).
(11-13). Protein concentrations were determined by the procedure of r3H]NaBH4 and uniformly labeled [14C]uridine were purchased from New England Nuclear. NADPH, ATP, and dithiothreitol were purchased from Sigma Chemical Co. All other materials were purchased in the highest purity available.
Isolation of all compounds was accomplished using an Altex HPLC system with a Whatman ODS-1 column. All solutions were analyzed for radioactivity using Biofluor mixture and a Beckman model LS 7500 scintillation counter. ' The abbreviations used are: 2'-CIUDP, 2'-chloro-2'-deoxyuridine 5"diphosphate; HPLC, high performance liquid chromatography. dure of Cook and Moffatt (15). The 3"ketone (0.680g, 1.07 mmol) was placed in a 50-ml round bottom flask in 30 ml of absolute ethanol. [3H]NaBH4 (13 mg, (0.3 mmol) 100 mCi) was added to this solution and the reaction was allowed to proceed for 20 min at room temperature. At the end of the period, 600 mg (0.016 mol) of unlabeled NaBH., was added to the reaction vessel and the reaction proceeded for an additional 2 h at room temperature. The EtOH was removed in uacuo and the product was extracted with 3 x 50 mi of CHCL from 50 ml of H2O. The CHC13 layer was dried over MgS04, filtered, and concentrated in vacuo. In our hands, this procedure resulted in production of 65:35 mixture of 1-(2,5-di-0trityl-P-D-xylofuranosyl)uracil and 1-(2,5-di-O-trityl-P-D-ri-bofuranosy1)-uracil. The desired compound was isolated by a modification of the procedure described by Cook and Moffatt using CCb/acetone (5:l) and Merck Silica Gei G ( R~0 . 0 8 and 0.12, respectively). The compound was then repurified using TLC grade Silica Gel 60H column chromatography. The peak fractions were pooled and rechromatographed by column on the TLC grade silica gel.
The desired compound was then deblocked using 80% acetic acid described by Cook and Moffatt (15). The pmr spectrum showed uridine with no contaminating xylo isomer (specific activity, 8 X lo7 cpm/pmol).
The compound was then isotopically diluted 1:8 with unlabeled uridine and converted to the corresponding 5"mOnOphosphate by the procedure of Yoshikawa and Takenishi (18) and to the diphosphate by activation of the monophosphate with carbonyldiimidazole (19) (specific activity, 9.7 X lo6 cpm/ pmol). A second batch of [3'-"H]UDP prepared analogously had a specific activity of 9.1 X lo6 cpm/pmol. At each stage, the nucleotide was purified by DEAE-Sephadex G-25 chromatography by elution with the appropriate triethylammonium bicarbonate linear gradients (18,19).
[I4C]UDP was analogously synthesized from uniformly labeled uridine and had a specific activity of 5 X IO5 cpm/pmol.
T o each fraction, 50 p1 of 0.5 M Tris (pH 8.5) and 5 p1 of alkaline phosphatase E. coli (1.75 units) were added and incubated for 1 h at 37°C. The samples were then boiled for 1 min and the protein was removed by centrifugation. The supernatant was removed with a disposable pipette and placed in a 5-ml round bottom flask. The protein precipitate was washed with 0.845 mi of H20 and the H20 wash was placed in the same flask. The H20 was then removed by bulb to bulb distillation.
The dried residue was redissolved in 100 pl of H20 and injected directly onto the Whatman ODS-1 reverse phase column (flow, 1.7 ml/min, H 2 0 eluant) and 0.4-min fractions collected. Compounds and retention times: uridine, 3.25 min; deoxyuridine, 5.5 min. The uridine and deoxyuridine peaks were purified to constant specific activity by HPLC. All specific activities are reported in counts per min/pmol, as d samples were counted with 48% efficiency. Extent of reaction was determined by weighing the appropriate HPLC traces.
The specific activities of uridine and deoxyuridine were determined by counting a known amount of sample ( A, , , 260, E = 1 X io4 M-' cm").
After the initial isolation by HPLC of the products from each individual time point, the appropriate uridine and deoxyuridine peaks were pooled and recounted. "H/I4C windows were set such that 20,000 cpm in the "H channel overlapped 1.2% into the I4C channel and 5,000 cpm in the I4C channel overlapped 11% into the ,"H channel. A typical specific activity of uridine or deoxyuridine was determined with 20,000 counts in the 'H channel.
2) Protocol was identical with that described in 1) except that solutions were not treated with alkaline phosphatase. Instead, UDP and dUDP were separated by ion pair reverse phase chromatography directly on the Whatman reverse phase ODS-1 column using an isocratic elution with 0.055 M KP (pH 5.5) and 5 mM tetrabutylammonium hydroxide, flow rate, 1.5 ml/min. Compounds and retention times: UDP, 4.5 min; dUDP, 7.0 min. A late migrating impurity was observed (retention time, 10 to 12 min) accounting for 1.5% of [3'-,"H]-UDP and 5.6% of [I4C]UDP.

RESULTS
Three independent methods were used to determine the "H selection effect on the conversion of UDP to dUDP catalyzed by ribonucleoside diphosphate reductase. These methods are: 1) single label experiment using [3'-'H]UDP and analysis of uridine and deoxyuridine (Fig. lA), 2) a double label experiment using [3'-3H]UDP/[14C]UDP and analysis of uridine and deoxyuridine (Table I), and 3) a double label experiment using [3'-3H]UDP/['4C]UDP and analysis of UDP and dUDP (Table I). Methods l and 2 provide more accurate determination of the specific activity of uridine during 5 to 50% conversion to product. Method 3 provides more accurate determination of the specific activity of uridine at >70% reaction. Errors involved with each method are discussed subsequently. 1) Single Label (3'-3H/UD~-[~-"H]UDP was incubated with ribonucleoside diphosphate reductase for fixed time intervals. Aliquots of the reaction mixture were treated with   indicate that the specific activity of deoxyuridine is clearly less than the specific activity of starting uridine. For example, at 5% reaction, the specifk activity of deoxyuridine is 2.5 X lo6 cpm/pmol, while that of uridine is 9.6 X lo6 cpm/pmol. This observed difference in specific activity of starting material and product at early time points indicates a selection against ['HIUDP during enzyme-catalyzed reduction. Fig. lA indicates that this selection against 3H is approximately 3.8. Also indicated in Fig. lA is that the specific activities of both uridine and deoxyuridine increase with the extent of the reaction. The increase in specific activity of uridine with time is a reflection of the selection against [3H]UDP during reduction. The increase in specific activity of the deoxyuridine is a reflection of the increased specific activity of uridine. Furthermore, at 100% reaction, the specific activity of deoxyuridine should be identical with that of the starting material if no 3H is lost during this reduction. As indicated in Fig. l A , the specific activity of deoxyuridine at 90% reaction is 7.5 X lo6 cpm/pmol and approaches that of the starting material, which is 9.1 x lo6 cpm/pmol. The greatest inaccuracy of this single label method for determining the selection against [3H]UDP involves measurement of the specific activity of uridine at greater than 70% conversion to products. Decreased uridine specific activity is due to ultraviolet absorbing materials and radiolabeled materials which migrate with similar retention times to uridine upon HPLC analysis. These impurities result from several sources. 1) [3'-"H]UDP is contaminated with an unknown phosphorylated uridine (1.5 to 3.2% depending on the [3H]-UDP preparation) which migrates with a retention time of 10 to 12 min on ion pairing reverse phase column chromatography. This impurity, when treated with alkaline phosphatase, resulted in a compound which migrated in the uridine region on analysis by reverse phase chromatography, Hz0 eluant. 2) Uridine has a short retention time (3.5 min) by HPLC analysis and thus migrates near the void volume of the column (2 to 2.5 ml). Small amounts of UV absorbing material (~5 % ) eluted in the void volume, therefore, may contaminate the isolated uridine. 3) Analysis of the specific activity of uridine at >90% reaction is complicated by the fact that 3H20 is released during the enzyme-catalyzed reduction (Fig. 1B). It  increasing the uncertainty of the specific activity of uridine at late times, are not a problem at early times.
Results from this same single label experiment indicated that the reduction reaction catalyzed by ribonucleoside diphosphate reductase is accompanied by release of a small but significant amount of 'H20 ( Fig. le). At 5% reaction, when we observe a selection against [3H]UDP of -3.8, only a very small amount of total counts (0.003%) has been released as "H20. Thus, under initial reaction conditions, the selection effect should not be significantly altered by " 2 0 release. At late reaction times where, at 80% reaction, 1.8% of total counts have been volatilized, analysis of specific activity of uridine becomes more complicated.
Because the percentage of counts volatilized was small compared with the total counts in each experiment, control experiments with heat-inactivated ribonucleoside diphosphate reductase were run under identical conditions. In a typical experiment containing 4.6 X lo6 cpm, at 85% reaction, 8.8 X lo4 cpm volatilized in the experiment was accompanied by 2.1 x lo3 cpm (0.045% total counts) volatilized in the control. These background counts remained constant with time. Each time point recorded in Fig. 1B has been corrected for this background.
This 'HzO formation also reflects the selection against [3H]UDP. If there were no selection effect on cleavage of the 3°C-H bond, a linear relationship would be expected between extent of reaction and percentage of 3H20 released. The fact that the 'HZO release deviates from linearity with the extent of reaction reflects the observed selection against ['HIUDP (Fig. lA).
2) Double Label Experiment I-To avoid the problems with UV absorbing impurities, [3"3H]UDP and [14C]UDP were incubated with ribonucleoside diphosphate reductase and the reaction mixture was analyzed for uridine and deoxyuridine. The results of this experiment are indicated in Table la. At 7% reaction, a selection against [3H]UDP of 3.3 is observed. This reduction reaction is also accompanied by 3H20 release. No 14C material is volatilized. The release of 'HzO as a function of extent of reaction is similar to that described in the single label experiment (data not shown).
Analysis of this double label experiment by HPLC revealed that [3H]uridine and [3H]deoxyuridine eluted from the reverse phase column slightly ahead of the corresponding 14C-labeled nucleosides. Appropriate fractions were therefore pooled and