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Biochemistry 2003, 42, 4578-4584
Epimerization at Carbon-5′ of (5′R)-[5′-2H]Adenosylcobalamin by Ribonucleoside Triphosphate Reductase: Cysteine 408-Independent Cleavage of the Co-C5′ Bond† Dawei Chen,‡ Andreas Abend,‡ JoAnne Stubbe,§ and Perry A. Frey*,‡ UniVersity of Wisconsin-Madison, 1710 UniVersity AVenue, Madison, Wisconsin 53726, and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed January 15, 2003; ReVised Manuscript ReceiVed January 31, 2003
ABSTRACT:
The adenosylcobalamin-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of ribonucleoside triphosphates to deoxyribonucleoside triphosphates. RTPR also catalyzes the exchange of the C5′-hydrogens of adenosylcobalalamin with solvent hydrogen. A thiyl radical located on Cys 408 is generated by reaction of adenosylcobalamin at the active site and is proposed to be the intermediate for both the nucleotide reduction and the 5′-hydrogen exchange reactions. In the present research, a stereochemical approach is used to study the mechanism of the CoC5′ bond cleavage of adenosylcobalamin in the reaction of RTPR. When stereoselectively deuterated coenzyme, (5′R)-[5′-2H1] adenosylcobalamin (5′R/S ) 3:1), was incubated with RTPR or the Cys 408 viariants, C408A-RTPR and C408S-RTPR in the presence of dGTP, the deuterium at the 5′-carbon was stereochemically scrambled, leading to epimerization of the (5′S)-[5′-2H1]- and (5′R)-[5′-2H1]-isotopomers. Observation of epimerization with mutated RTPR proves that transient cleavage of the Co-C5′ bond occurs in the absence of the thiol group on Cys 408. The rate constants for epimerization by RTPR, C408A-RTPR, and C408S-RTPRs in the presence of dGTP are 5.1, 0.28, and 0.42 s-1, respectively. Only the wild-type RTPR catalyzes the 5′-hydrogen exchange reaction. Both epimerization and 5′-hydrogen exchange reactions are stimulated by the allosteric effector dGTP, and epimerization is not detected in the absence of the effector. Mechanistic implications with respect to wt-RTPR-mediated carbon cobalt bond homolysis and the intermediacy of the 5′-deoxyadenosyl radical will be presented.
In vivo, deoxyribonucleotides are produced by reduction of ribonucleotides in reactions catalyzed by ribonucleotide reductases. The Lactobacillus leichmannii ribonucleoside triphosphate reductase (RTPR1), a class II ribonucleotide reductase, requires adenosylcobalamin and catalyzes the reaction shown in eq 1 in which reduction is accompanied by active site disulfide formation (1). † Supported by Grant DK 28607 from the National Institute of Diabetes and Digestive and Kidney Diseases (P.A.F.) and GM.29595 from the National Institute of General Medical Sciences (J.S.). This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH Grant RR02301 from the Biomedical Research Technology Program, National Center for Research Resources. Equipment in the facility was purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program (Grant DMB-8415048), NIH Biomedical Research Technology Program (Grant RR02301), NIH Shared Instrumentation Program (Grant RR02781), and the U.S. Department of Agriculture. * Corresponding author. Tel: (608) 262-0055. Fax: (608) 265-2904. E-mail:
[email protected]. ‡ University of Wisconsin-Madison. D.C. and A.A. contributed equally to this work. § Massachusetts Institute of Technology. 1 Abbreviations: RTPR, ribonucleoside triphosphate reductase; C408A-RTPR, C408A variant of ribonucleoside triphosphate reductase; C408S-RTPR, C408S variant of ribonucleoside triphosphate reductase; adenosylcobalamin, 5′-deoxyadenosylcobalamin; dGTP, 2′-deoxyguanosine 5′-triphosphate; ATP, adenosine 5′-triphosphate; NADPH, β-nicotinamide adenine dinucleotide; DTT, dithiothreitol; TR, thioredoxin; TRR, thioredoxin reductase; D2O, deuterium oxide; 1H NMR, proton nuclear magnetic resonance spectroscopy; TFA, trifluoroacetic acid.
Thioredoxin (TR) is essential for multiple turnovers, rereducing the active site thiols indirectly by disulfide interchange through two additional C-terminal thiols (2, 3). RTPR catalyzed reduction also requires deoxyribonucleoside triphosphates and/or ATP as allosteric effectors to control the rate of reduction and the substrate specificity (4). RTPR is unique among adenosylcobalamin requiring proteins in that in addition to the reduction reaction, it catalyzes the exchange of 5′-methylene hydrogens of adenosylcobalamin with solvent (5, 6). The rate constant for the exchange reaction is similar to that of nucleotide reduction (7). The exchange reaction also requires the presence of an allosteric effector, and both reactions require adenosylcobalamin dependent formation of a transient thiyl radical. Rapid freeze quench EPR experiments have identified the transient thiyl radical and shown it to be kinetically competent (8, 9). Site-directed mutagenesis experiments, and more recently crystallographic studies, have identified C408 as the locus of this protein radical (3, 11). The role of adenosylcobalamin is thus unique relative to other B12
10.1021/bi030018x CCC: $25.00 © 2003 American Chemical Society Published on Web 03/28/2003
Co-C Bond Cleavage Ribonucleoside Triphosphate Reductase requiring proteins; its role is to generate a transient protein radical, not a carbon centered radical on a substrate. In recent presteady state kinetic studies, which were based on the original experiments of Blakley and Tamao (12), Stubbe and co-workers proposed that both the exchange reaction and the reduction reaction proceed through a common intermediate: a thiyl radical, cob(II)alamin, and 5′deoxyadenosine (7, 8). Analysis of the kinetics and isotope effects on the exchange reaction led to the proposal that formation of this intermediate from adenosylcobalamin and C408-RTPR occurred in a concerted fashion, eq 2 (7). However, an alternative stepwise mechanism (eq 3) in which a high energy 5′-deoxyadenosyl radical intermediate and cob(II)alamin is generated followed by rapid formation of thiyl radical and 5′-deoxyadenosine could not be excluded.
The present work was undertaken in a search for the 5′deoxyadenosyl radical intermediate and to better understand the mechanism of Co-C5′ bond cleavage in the reaction catalyzed by RTPR. EXPERIMENTAL PROCEDURES Materials. Adenosylcobalamin, dGTP, ATP, β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), dithiothreitol (DTT), TR, and thioredoxin reductase (TRR) were purchased from Sigma. Deuterium oxide (D2O, 99.9 atom % D) was from Aldrich. HPLC grade methanol was from Fisher Scientific. Microcon-30 centrifugal filter devices were obtained from Millipore. The filter devices employ Amicon low-binding, hydrophilic regenerated cellulose membranes of low absorption characteristics. SepPak C18 cartridges were from Waters. Eschericia coli cells (strain HB101) transformed with the expression plasmid carrying the RTPR gene from L. leichmannii and E. coli cells (strain BL21) transformed with the expression plasmid carrying the genes for expression of C408S-RTPR and C408A-RTPR were as described (3, 10). Synthesis of StereoselectiVely Deuterated [5′-2H] Adenosylcobalamin. Synthesis of (5′R)-[5-2H1]adenosylcobalamin [(5′R)/(5′S) ) 3:1] was carried out in the dark as described elsewhere (13). Stereochemical analysis by 1H NMR showed it to consist of a 3:1 mixture of 5′R/5′S epimers. This material is hereafter referred to as (5′R)-[ 5′-2H1]adenosylcobalamin. The product was purified in the dark by reverse-phase chromatography on a Superformance glass cartridge (2.5 × 30 cm, E. Merck, Germany, packed with octadecyl functionalized silica gel from Aldrich, equilibrated in 0.02% trifluoroacetic acid (TFA)/water). The reaction mixture was acidified with concentrated TFA to pH 1, diluted to 150 mL, and transferred into a 150 mL Super-Loop (Pharmacia, Sweden). The Super-Loop was connected to a FPLC system (Pharmacia, Sweden), and the sample was loaded at a flow
Biochemistry, Vol. 42, No. 15, 2003 4579 rate of 5 mL/min. The loop was removed from the system, and the column was washed with 200 mL of 0.02% TFA (A). A linear gradient starting at 100% A to 75% B (80% methanol, 0.02% TFA) was applied at 5 mL/min and over 220 min. Fractions (20 mL) were collected, and aliquots were examined by UV/Vis spectroscopy. (5′R)-[5-2H1]Adenosylcobalamin emerged at 28% B. Pure fractions containing (5′R)-[5-2H1]adenosylcobalamin were pooled, evaporated under reduced pressure, and redissolved in 5 mL of water. The desired product was absorbed on a SepPak C18 cartridge (3 mL, Waters), and the cartridge was washed with water until the flow-through was neutral. (5′R)-[5-2H1]Adenosylcobalamin was then eluted with methanol. The (5′R)-[52 H1]adenosylcobalamin in methanol was diluted to 1.5 mg/ mL, and 1 mL aliquots were evaporated in vacuo in amber 1.5 mL centrifuge tubes and stored at -20 °C. General Methods. Solution concentrations were measured spectrophotometrically using the following molar extinction coefficients: 280 of 101 000 M-1 cm-1 for RTPR (1), 525 ) 8000 M-1 cm-1 for adenosylcobalamin (14), and 260 ) 15 400 M-1 cm-1 for dGTP and ATP (15). All experiments involving adenosylcobalamin were carried out in dark or under dim light. In D2O solutions, all measured pHs were corrected to pD by addition of 0.4 to the reading. Enzyme activities were assayed by the coupled assay method using TRR/TR/NADPH as the reducing agents (1). The assay mixture (1.0 mL) contained 50 mM HEPES (pH 7.4), 1.0 mM dGTP, 2.0 mM ATP, 200 µM NADPH, 12 µM TR, 0.16 µM TRR, and 0.5 µM RTPR or 10 µM C408A-RTPR. The reaction was initiated by the addition of adenosylcobalamin (5.0 µM), and the decrease in A340 nm was monitored. Background was corrected by running a control reaction in the absence of the added enzyme. Rates were calculated using ∆340 ) 6220 M-1 cm-1. A specific activity of 0.41 ( 0.05 µmol min-1 mg-1 (kcat ) 0.55 ( 0.07 s-1) was measured at 37 °C. No activity for C408A-RTPR was detected. Measurement of Adenosylcobalamin Binding to C408ARTPR. Measurement of adenosylcobalamin binding was carried out by ultrafiltration using Microcon-30 centrifugal filter devices (Millipore) and a 30 K cutoff filter as described (16). In one set of experiments, the solution contained 50 mM HEPES (pH 7.4), 105 µM adenosylcobalamin, 2.0 mM dGTP, 20 mM DTT, and from 40 to 520 µM of C408ARTPR in a total volume of 500 µL. The mixtures were transferred to the filter devices after being incubated at 37 °C for 10 min and centrifuged for 3-5 min so that less than 15% of the solution had passed into the filtrate. Controls excluding protein revealed that the amount of adenosylcobalamin bound to the filters was small (
