Characterization of a Nucleotide Kinase Encoded by Bacteriophage T7*

Background: Gene 1.7 of bacteriophage T7 encodes a nucleotide kinase. Results: gp1.7 dodecamers catalyze the reversible dTMP and dGMP kinase reactions. Conclusion: gp1.7 is a unique nucleotide kinase that does not require a divalent metal ion. Significance: The unique nucleotide kinase of bacteriophage T7 supplies dTDP and dGDP for conversion to the nucleoside 5′-triphosphate and recycles the dTDP derived from helicase function to dTTP. Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ∼12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg2+ in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The Km of 4.4 × 10−4 m obtained with dTTP for dTMP kinase is ∼3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10−4 m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.

Gene 1.7 of bacteriophage T7 came to our attention when we found that mutations in gene 1.7 rendered T7 growth on Escherichia coli resistant to exogenous dideoxythymidine (ddT) in the media (1). E. coli can grow in the presence of ddT up to 5 mM, whereas T7 fails to form plaques in the presence of 0.1 mM ddT. Rare ddT-resistant phage did appear, and essentially all of them had a mutation in gene 1.7. Although genetic data sug-gested that gene 1.7 protein (gp1.7) 2 was involved in nucleotide metabolism, it was not until the protein was purified that it was identified as a nucleotide kinase that phosphorylates both dTMP and dGMP to dTDP and dGDP, respectively (2). One of the interesting properties of the T7 nucleotide kinase is that it phosphorylates ddTMP with essentially the same efficiency as it does dTMP. We have shown that the host E. coli thymidylate kinase (EC 2.7.4.9), whose activity on dTMP is comparable with the T7 gp1.7, discriminates against the use of ddTMP by more than 500-fold (2). This lack of specificity reveals the mechanism for selective inhibition of phage T7 growth by ddT (2); ddT enters E. coli cells and is phosphorylated to ddTMP by E. coli thymidine kinase (EC 2.7.1.21). The conversion of ddTMP to ddTDP by gp1.7 maintains this chain-terminating nucleotide on the pathway to ddTTP, which is readily incorporated into T7 DNA by T7 DNA polymerase (3). Finally, the incorporation of ddTMP into DNA results in termination of the chain and thus inability of the phage genome to be successfully replicated.
In E. coli cells infected with bacteriophage T7, synthesis of T7 DNA occurs at a rapid rate. T7 DNA synthesis is initiated between 5 and 10 min after infection and reaches a maximum rate between 15 and 20 min after infection. The rate of T7 DNA synthesis is ϳ5-10-fold the preinfection rate of E. coli DNA synthesis (4,5). This rapid DNA synthesis requires a large pool of deoxynucleoside 5Ј-triphosphate (dNTP) precursors for T7 DNA polymerase. In addition, during DNA replication the unwinding of the DNA by the T7 helicase is fueled by the hydrolysis of dTTP (6). T7 has bypassed a rate-limiting step in the synthesis of the dNTP precursors by using the deoxynucleoside monophosphates obtained from the breakdown of the host DNA (7,8), thus bypassing the complex de novo reduction of ribonucleotides to deoxyribonucleotides. This degradation is accomplished by the combined action of the T7 gene 3 endonuclease (9, 10) and gene 6 exonuclease (11).
The identification of the T7 nucleotide kinase that phosphorylates dTMP (EC 2.7.4.9) and dGMP (EC 2.7.4.8) to the corresponding dTDP suggests that the E. coli nucleotide kinases are not sufficient to provide an adequate supply of dNTPs for the * This work was supported by United States Public Health Service Grant GM54397. 1  synthesis of T7 DNA. No other T7 phage-encoded nucleotide kinase has been identified. It would appear that the E. coli cytidylate kinase (EC 2.7.4.14) is responsible for the conversion of dCMP to dCDP in T7-infected cells as this enzyme is essential for T7 growth (12). Aside from its interesting role in T7 DNA replication, the T7 nucleotide kinase is a fascinating enzyme. gp1.7 does not share sequence homology with any known nucleotide kinase, and there are no identifiable nucleotide binding motifs found in its protein sequence. A most unusual feature is its full activity in the absence of added metal ion (2). gp1.7 exists as two molecular mass forms of 22 and 18 kDa. The smaller form is missing the first 41 residues at the N terminus found in the larger form as a result of an internal ribosome-binding site and an in-frame start codon within the coding sequence (2). The physical properties of the protein are also quite unusual. T7 gp1.7 is precipitated by the presence of even small amounts (50 mM) of NaCl. This curious property has facilitated its purification by sequential NaCl precipitation and resolubilization steps. This communication describes the further characterization of this unique nucleotide kinase with a focus on the distinct mechanisms by which the enzyme regulates deoxyguanosine and thymidine substrates.

EXPERIMENTAL PROCEDURES
Purification of T7 Nucleotide Kinase by Ni-NTA Affinity Chromatography-Full-length gene 1.7 protein (gp1.7) fused with a His 6 tag at its N terminus was expressed as previously described (2). Cells from 6 liters of culture were lysed by sonication in binding buffer (50 mM Tris-Cl, pH 7.5, 1 mM PMSF, 10 mM ␤-mercaptoethanol, 0.1% Tween 20, 10% glycerol) followed by centrifugation. The following components were added to the supernatant: 300 mM NaCl, 50 mM imidazole, 3 ml of Ni-NTA agarose resin. Note that in the presence of 300 mM NaCl, gp1.7/ His 6 binds tightly to Ni-NTA even in the presence of 50 mM imidazole, thus minimizes nonspecific binding. After allowing binding to proceed for 2 h at 4°C, the mixture was poured into a column (10 ϫ 1.5 cm). The column was washed sequentially with: (i) 100 ml of binding buffer containing 300 mM NaCl and 50 mM imidazole; (ii) 50 ml of binding buffer containing 300 mM NaCl and 100 mM imidazole; (iii) 100 ml of a linear gradient of NaCl (300 to 0 mM) in buffer containing 20 mM Tris-Cl pH 7.5, 10 mM ␤-mercaptoethanol; and (iv) finally with 50 ml of buffer containing 20 mM Tris-Cl, pH 7.5, 10 mM ␤-mercaptoethanol. Bound protein was eluted by steps of 0.1, 0.3, and 0.5 M imidazole in buffer containing 20 mM Tris-Cl, pH 7.5, and 10 mM ␤-mercaptoethanol. The protein was further purified by gel filtration using Sephacryl S-400 HR column as previously described (2).
Purification of gp1.7/His 6 under denaturing conditions followed by renaturation of the protein on the column was carried out using conditions modified from those previously described (13). After binding step, the resin was slowly washed with the following solutions: (i) 10 column volumes (cv) of 8 M urea, 20 mM Tris-Cl, pH 8, 10 mM ␤-mercaptoethanol; (ii) 10 cv of Buffer A (20 mM Tris-Cl, pH 8, 100 mM NaCl, 10 mM ␤-mercaptoethanol) containing 0.1% Triton X-100; (iii) 10 cv of Buffer A containing 5 mM ␤-cyclodextrin and 10 mM imidazole; and (iv) 10 cv of Buffer B (20 mM Tris-Cl, pH 7.5, and 10 mM ␤-mercaptoethanol). Protein was eluted with the Buffer B containing 0.3 M imidazole.
Molecular Weight Determination-The native molecular weight of purified gp1.7 was determined by multiple-angle light scattering (MALS). Purified gp1.7 was flowed through a Sephadex-75 gel filtration column using an AKTA-FPLC system (GE Healthcare) connected online to a MALS system, DAWN HELEOS TM II (Wyatt Technology Corp.). Operation of the system and interpretation of the results was followed as in the manufacturer's instructions. The purified gp1.7 was also examined by electron microscopy (EM). The EM samples were prepared by resuspending the protein in buffer containing 20 mM Tris-Cl, pH 7.5, 10 mM ␤-mercaptoethanol, and 50 M dTMP. Samples were adsorbed to glow-discharged, carbon-coated EM grids and stained with 0.75% (w/v) uranyl formate solution as described previously (14). Images were collected using Tecnai TM  H]dTMP, and 500 ng of E. coli thymidylate kinase that was purified as previously described (2). After incubation at 37°C for 30 min, the reaction was stopped by heating at 95°C for 3 min. The mixture was diluted two times with water and applied to a DEAE DE52 cellulose column (1.6 ϫ 2 cm) pre-equilibrated with 50 mM NH 4 HCO 3 , pH 7.8. The column was washed with 10 ml of 50 mM NH 4 HCO 3 , pH 7.8. Bound [ 3 H]dTDP was eluted by a 100-ml linear gradient (50 -300 mM) of NH 4 HCO 3 pH 7.8. 1-ml fractions were collected, and an aliquot was checked for radioactivity.
[  3 ]dTMP (ϳ10 cmp/pmol), 5 mM dTTP and the indicated amounts of gp1.7. Reactions were carried out at 37°C for the indicated times and terminated by heating the mixture at 95°C for 3 min. The mixture was separated along with a marker containing 1 g of each unlabeled dTMP, dTDP, and dTTP by PEI-cellulose TLC in the solvent consisting of 0.5 N LiCl and 2 N acetic acid. In this solvent dTMP, dTDP, and dTTP migrate with R f values of 0.8, 0.4, and 0.04, respectively. The nucleotides were localized under UV light at 254 nm and cut out, and radioactivity was determined by liquid scintillation counting. dGMP kinase assays were per-formed in an identical procedure with deoxyguanosine substrates.
Reversibility of the dTMP kinase reaction was determined by measuring the amount of [  Inductively Coupled Plasma Mass Spectroscopy Analysis of Metal Content-Inductively coupled plasma mass spectroscopy analysis was performed at Trace Metal Laboratory, Harvard School of Public Health, Boston, MA based on the procedure previously described (15). Three different forms of gp1.7 were analyzed: the wild-type enzyme (a complex of the two molecular weight forms), the 22-kDa species, and the 18-kDa species. These protein samples did not contain a histidine tag and were purified by salting in/out as described (2).

Co-purification of Two Molecular Weight Forms and General
Properties of gp1.7-gp1.7 exists in two molecular mass forms of 18 and 22 kDa. The smaller form that lacks the first 41 N-terminal residues arises as a result of an internal ribosome-binding site and in-frame initiation site (2). T7 gp1.7 has the interesting property of precipitating in the presence of even low concentrations of NaCl. We have previously reported the co-purification of the two forms of gp1.7 by serial salting in/out with NaCl (2). However, this method is time-consuming and results in considerable losses with each precipitation and resolubilization. In this study we describe an effective and simpler method to purify this enzyme. We overproduced gp1.7 fused to a His 6 tag at its N terminus and purified the enzyme using Ni-NTA affinity chromatography and gel filtration (see "Experimental Procedures"). By attaching the His 6 tag to the N terminus of the full-length gp1.7, we also anticipated that Ni-NTA affinity chromatography would result in the purification of only the 22-kDa form, as the18-kDa form of gp1.7 does not contain the His tag and thus in theory would not bind to the Ni-NTA resin. Surprisingly, the 18-kDa species co-purified on the Ni-NTA affinity with the 22-kDa gp1.7 (Fig. 1A, lanes 3 and 4). When gp1.7 was purified under denaturing conditions using Ni-NTA affinity, no 18-kDa species was detected (Fig. 1A, lane 5 and 6). This result indicates that the two species interact physically to form a complex; the 18-kDa species binds to the 22-kDa/His tag protein and thus co-purifies with it.
Purification of gp1.7 using His tag followed by gel-filtration resulted in nearly homogeneous gp1.7 as seen on SDS-PAGE stained with Coomassie Blue (Fig. 1A, lane 4). Separate experiments showed that gp1.7/His tag and wild-type gp1.7 purified by serial NaCl salting in/out exhibit the same specific activity for conversion of dTMP to dTDP (data not shown). Fig. 1B shows that the 18-and 22-kDa species each purified alone and wild-type (wt) gp1.7 containing an equimolar mixture of the two species had essentially identical kinase activities with respect to the conversion of dTMP to dTDP. Therefore, wildtype gp1.7 with His tag, unless otherwise indicated, was used for routine assays described in this study. gp1.7 exhibits an optimum activity in the pH range of 7-7.5 in Tris-Cl buffer ( Fig. 2A). The sensitivity of the kinase to temperature is shown in Fig. 2B. In this experiment the enzyme was incubated at the indicated temperatures for 5 and 10 min before immediately assaying in the standard reaction at 37°C for 2 min. The activity was nearly unaffected by temperatures up to 40°C but rapidly declined at higher temperatures; ϳ50% activity remained after 10 min at 45°C, whereas Ͻ1% of the activity remained after incubation for 10 min at 65°C.
The solubility of gp1.7 in varying concentrations of NaCl and KCl is presented in Fig. 2C. In this experiment increasing FIGURE 1. Purification and comparison of kinase activity of the two molecular weight forms of gp1.7. A, expression and purification gene 1.7 protein is shown. Overproduced gp1.7/His tag was purified as described under "Experimental Procedures." Lanes 1 and 2, lysate from uninduced and induced cells, respectively; lanes 3 and 4, purified gp1.7 after Ni-NTA affinity chromatography and gel filtration, respectively; lane 5, gp1.7 that was denatured, purified using Ni-NTA affinity chromatography, and renatured; lane 6, the renatured protein following by gel filtration. B, comparison of kinase activity of wild-type (E), 22-kDa (F), and 18-kDa (OE) gp1.7 is shown. Each gp1.7 species was purified by salting in/out as described previously (2) amounts of either salt were added to a solution containing gp1.7. The precipitated gp1.7 was removed by centrifugation, and the supernatant were collected and used for the assay. The % activity reflects the amount of soluble gp1.7 remaining in supernatant as compared with no salt treatment (no precipitation). Approximately 70% of gp1.7 precipitates at 50 mM NaCl, whereas essentially all (Ͼ95%) is precipitated by the presence of 100 mM NaCl as measured by the activity remaining in the supernatant after centrifugation. Essentially identical results were obtained with KCl (Fig. 2C). In each case all of the activity not in the supernatant can be recovered from the pellet by dissolving the protein in buffer lacking NaCl. Interestingly, although both NaCl and KCl precipitate gp1.7, neither salt is inhibitory to kinase activity when added to the standard reaction mixture up to 500 mM (Fig. 2D). Based on these properties, the standard assays of gp1.7 in this study were carried out at 37°C in Tris-Cl buffer pH 7.5 without salt.
T7 gp1.7 Forms Dodecamers-The purification profile discussed above suggests that gp1.7 interacts with itself to form oligomers. The Stokes radius of gp1.7 was analyzed in the absence of NaCl by gel filtration on HiPrep 26/60 Sephacryl S-200HR (fraction range, 5-250 kDa) using an AKTA-FPLC. Results presented in Fig. 3A show that gp1.7 elutes in the excluded volume in the same position as apoferritin (449 kDa) and thyroglobulin (669 kDa).
We further analyzed gp1.7 by electron microscopy (Fig. 3B). The majority of particles have a globular shape with an average diameter of 25.6 Ϯ 0.8 nm, much larger than the expected diameter of a gp1.7 monomer. However, the resolution is not sufficient to determine the stoichiometry of the gp1.7 complexes.
We used MALS to obtain a more precise estimate of the molecular mass of gp1.7. The preparation of gp1.7 contains an equimolar mixture of the two molecular weight forms, as shown in Fig. 1, lane 4. This analysis estimates the molecular mass of gp1.7 to be 256 kDa, equivalent to a complex containing ϳ12.2 monomers of gp1.7 based on equal amounts of the two molecular weight forms in the complex. Consistent with this result, a comparable MALS analysis of the 22-kDa gp1.7/His tag alone (ϳ24 kDa including the His tag) estimated the molecular mass to be 273 kDa, equivalent to ϳ11.3 monomers of the 22-kDa gp1.7/His tag. These results taken together suggest that the gp1.7 exists predominantly as a dodecamer. gp1.7 Activity Does Not Require Divalent Cations-We previously showed that T7 gp1.7 has kinase activity in the absence of added divalent cations even in the presence of the chelating agent EDTA (2). One possibility is that a divalent cation is required but that it is tightly bound to gp1.7 and not accessible to chelating agent. To explore this possibility, we analyzed the metal content of purified gp1.7 by inductively coupled plasma mass spectroscopy. Three different preparations of gp1.7 were analyzed: the 22-kDa, the 18-kDa, and the wild-type species consisting of both molecular weight forms. In each case the proteins did not contain His tag and were purified by serial salting in/out (2). Initially, 21 divalent cations were screened. Based on these results, the four cations that gave the strongest signals were further analyzed for their content (Table 1). Mg 2ϩ , the divalent cation most commonly used in kinase reactions, is found in only a 1:30 molar ratio with gp1.7 monomers. Only zinc was present at a significant level and only in the 22-kDa and wild-type species; the highest level of this metal was ϳ0.3 mol/ mol of protein (Table 1). gp1.7 has nine cysteines, seven of which are located within the first 79 N-terminal residues. These cysteines are found in two sequences: 21 CX 2 CX 10 C 35 and 67 CX 3 CX 4 CX 2 C 79 that are putative zinc binding motifs (16). Therefore, it is likely that these cysteines account for binding the detected zinc. Consistent with this interpretation, the 18-kDa gp1.7 that lacks the first 41 N-terminal residues contains 10-fold less zinc: 0.04 mol/mol of gp1.7 monomer (Table 1). However, the 18-kDa protein had the same kinase activity as does the 22-kDa species (Fig. 1B). Furthermore, deletion genetic mapping has shown that the N-terminal half of gp1.7 is not required for conferring sensitivity of T7 phage to dideoxythymidine (1,2). We have also purified gp1.7 under denaturing conditions followed by renaturation to release any tightly bound metal. The renatured gp1.7 had ϳ60% that of the kinase-specific activity as the protein purified under native conditions (data not shown). These results taken together strongly suggest that no divalent cations are required for activity of T7 gp1.7. A, shown is determination of the pH optima. dTMP kinase reactions were carried out in Tris-Cl buffers ranging in pH from 6 to 9. B, shown is the effect of temperature on dTMP kinase activity of gp1.7. 50 ng of gp1.7 was incubated in 20 mM Tris-Cl, pH 7.5, 1 mM DTT at the indicated temperatures for 5 (F) or 10 (‚) min before the addition to the standard reaction mixtures. Reactions were carried out at 37°C for 2 min. Activities are compared with a standard reaction carried out without prior incubation. C, shown is precipitation of gp1.7 by NaCl (F) and KCl (‚). Varying concentrations (0 -750 mM) of either salt was added to the cell lysate containing overexpressed gp1.7 and then incubated on ice for 1 h. Samples were centrifuged at 14,000 rpm for 30 min using a microcentrifuge. Supernatants were collected and used for kinase assay in the absence of Mg 2ϩ . Activities were compared with a sample that was treated under identical conditions without salt. D, shown is the effect of NaCl (F) and KCl (‚) on kinase activity of purified gp1.7. The indicated concentrations of NaCl or KCl were added to the reaction mixtures containing 50 ng of purified gp1.7. Reactions were carried out at 37°C for 2 min.
Phosphate Acceptor and Donor Specificity-In the experiments presented in Table 2, we examined the ability of a number of nucleoside monophosphates to serve as acceptors of the phosphate from dTTP in the presence and absence of MgCl 2 . gp1.7 specifically phosphorylates dTMP, ddTMP, and dGMP in the presence or absence of Mg 2ϩ . Surprisingly, dUMP, an effective phosphate acceptor of known thymidylate kinases (17)(18)(19), was only a poor substrate for gp1.7. The activity with dUMP was only 1 and 10% that observed with dTMP in the presence and absence of Mg 2ϩ , respectively. Other analogues, including thymidine, dideoxythymidine, and 3Ј-azido-3Ј-deoxythymidine 5Ј-monophosphate (AZT), are not phosphate acceptors from dTTP. It is noteworthy that Mg 2ϩ reduces the reaction with dGMP by ϳ40% but slightly stimulated the ability of dTMP and ddTMP to accept phosphate.
Various nucleoside triphosphates were also examined for their ability to donate the phosphate to dTMP and dGMP in the presence and absence of Mg 2ϩ (Table 3). gp1.7 uses ribo-GTP, dGTP, dITP, dTTP, dUTP, ddGTP, and ddTTP as phosphate donors. The other nucleoside triphosphates tested gave less than 1% of the activity obtained with dTTP. Again, despite the nonessential nature of Mg 2ϩ , the addition of Mg 2ϩ either stimulated or inhibited the kinase activity of gp1.7 depending on the donor ( Table 3). The presence of Mg 2ϩ significantly stimulated the phosphorylation of dTMP by most of active donors. Indeed a 3.5-fold stimulation was observed with the phosphorylation of dTMP by dGTP, making it the best phosphate donor. Mg 2ϩ also stimulated the phosphorylation of dGMP with dGTP and ddTTP as the donors. In particular, the ability of ddGTP to donate phosphate to both dGMP and dTMP was very low in the absence of Mg 2ϩ but was increased by 20-and 30-fold, respectively, by the presence of Mg 2ϩ . The presence of Mg 2ϩ inhibited the phosphorylation of dGMP by the donors, dITP, GTP, dTTP, and dUTP by 35, 35, 40, and 60%, respectively.
In view of these differing effects of Mg 2ϩ on the kinase reaction we examined the effect of Mg 2ϩ concentration on the four acceptor/donor combinations of thymidine and deoxyguanosine nucleotides (Fig. 4). In the absence of Mg 2ϩ , dGMP/dTTP was the most active combination. The initial rate of 13 pmol/ ng/min obtained with this combination was almost 4-fold higher than the dTMP/dGTP combination, which gave the lowest initial rate (3 pmol/ng/min). Mg 2ϩ stimulated the initial rate of the reaction containing either dTMP or dGTP up to 5 mM. Conceivably, the greatest stimulation (Ͼ3-fold) was obtained with combination dTMP/dGTP. The reaction rates of dTMP/dTTP and dGMP/dGTP were also increased by 30 and 90%, respectively, by the presence of 5 mM Mg 2ϩ . In sharp contrast, the rate of the reaction containing dGMP/dTTP decreased by 40% at this concentration of Mg 2ϩ (Fig. 4).  The metal content of three preparations of gp1.7 that were purified using serial salting in/out (2) were analyzed using inductively coupled plasma mass spectroscopy based on the procedure previously described (15 (Fig. 5). A Lineweaver-Burk plot (20) of the inhibition of the phosphorylation of dTMP by dGMP shows that the curves intersect on the y axis, indicating dGMP acts as a competitive inhibitor of dTMP (Fig. 5A). Likewise, a competitive relationship was detected between the two phosphate donors, dGTP and dTTP (Fig. 5B). These results indicate a competitive binding site for dTMP and dGMP and likewise a binding site for dTTP and dGTP.

Competitive Inhibition of dTMP by dGMP-We examined the inhibitory effect of dGMP on the conversion of [ 3 H]dTMP to [ 3 H]dTDP with dTTP as donor
Stoichiometry of dTMP and dGMP Kinase Reactions-We determined the stoichiometry of nucleoside mono-, di-, and triphosphates in the gp1.7 dTMP and dGMP kinase reactions. For each kinase reaction two parallel mixtures containing equivalent concentration (0.1 mM) of acceptor and donor were prepared. One mixture contained 3 H-labeled acceptor (dTMP or dGMP), whereas the other mixture contained 3 H-labeled donor (dTTP or dGTP). Changes in the amount of reactants and products over time were measured by TLC. The radioactive components found after the reaction had reached equilibrium (120 min) are shown in Table 4. The results show that the amount of dTDP formed in the reaction corresponds to the total amount of dTMP and dTTP lost (Table 4). Similarly, dGDP formed corresponds to the total amount of dGMP and dGTP lost (Table 4). From these data, we conclude that the stoichiometry of the kinase reactions is represented by the equations Interestingly, the results in Table 4 reveal two distinguishable equilibrium patterns between dTMP and dGMP kinase reaction. The dTMP kinase reaction reached equilibrium even   though dTTP and dTMP were still in a considerable 2-fold excess over the amount of dTDP formed (Table 4). In contrast, the dGMP kinase reaction reached equilibrium only after the dGDP concentration was considerably 2-fold higher than dGMP and dGTP ( Table 4). The apparent equilibrium constants (K eq ) for the dTMP and dGMP kinase reactions derived from these experiments are 0.07 and 0.85, respectively. The results imply that the product dTDP has a strong inhibition on its synthesis. The inhibition can be overcome by increasing the concentration of phosphate donor. The experiment presented in Fig. 6A shows an exponential conversion of dTMP to dTDP in a reaction containing 0.  (Table 5).
We examined the ability of the nucleoside triphosphate products dTTP and dGTP to serve as phosphate donors to dTDP and dGDP, respectively (Fig. 7B). An increasing amount (0 -1 mM) of dGTP and dTTP was correspondingly added to the reactions containing 0.   dGDP is a potent inhibitor of the conversion of [ 3 H]dGMP to [ 3 H]dGDP when either dGMP or dGTP is present in excess (Fig. 8A). In both cases the rate of [ 3 H]dGDP formation was inhibited ϳ84% by 0.4 mM dGDP. Surprisingly, inhibition of dTMP kinase by dTDP was considerably weaker than that obtained with dGDP on the dGMP kinase reaction (Fig. 8B). The rate of [ 3 H]dTDP formation in the reaction containing 0.1 mM [ 3 H]dTMP and 2.5 mM dTTP was inhibited only 20% by 0.4 mM dTDP (Fig. 8B, empty triangles). Inhibition by dTDP on the rate of conversion of [ 3 H]dTTP to [ 3 H]dTDP was 2-fold greater in the reaction containing a 25-fold excess of dTMP over [ 3 H]dTTP (0.1 mM) (Fig. 8B, filled triangles). These results suggest that the presence of high dTTP prevents dTDP from binding to the dTMP site.
Lineweaver-Burk plots (20) indicate a competitive relationship between dGDP and dGMP (Fig. 9A) as well as dGTP (data not shown). However Lineweaver-Burk plots (Fig. 9B) of the reciprocals of the initial rates against the reciprocals of dTMP concentration in the presence of dTDP as an inhibitor shows the intersection of the curves does not fall on either axis, indicating a mixed type of inhibition of dTMP kinase by dTDP.
Effect of dTTP and dGTP Concentration on the Reaction-The influence of dTTP and dGTP concentration on the rate of reaction is presented as a Michaelis-Menten plot in Fig. 10. At low concentrations (Fig 10, inset) of dTTP, the rate of the dTMP kinase reactions is not proportional to the donor concentration. On the other hand, the rate of the dGMP kinase reaction is proportional at even low concentrations of dGTP. The apparent Michaelis-Menten constants, K m calculated for dTTP and dGTP, are 4.4 ϫ 10 Ϫ4 and 1.3 ϫ 10 Ϫ4 M, respectively. These results suggest that unusual high dTTP is required to saturate the enzyme as compared with dGTP.

DISCUSSION
T7 gp1.7 was originally identified as a thymidylate kinase (EC 2.7.4.9) based on its ability to phosphorylate dTMP to dTDP (2). There are fundamental differences in the properties of this enzyme from those of other known thymidylate kinases found in E. coli (19), yeast (17), and mouse (18). First, it does not share sequence homology with these other thymidylate kinases and does not contain any of the identifiable motifs found in nucle-   otide binding domains. Second, the substrate specificity for both phosphate acceptor and donors of gp1.7 is different from that of any other known thymidylate kinase; it phosphorylates dGMP as well as dTMP, whereas it uses dTTP and dGTP as phosphate donors despite their structural differences. Finally, the lack of any requirement for a divalent cation for catalytic activity is unique among all known thymidylate kinases (17)(18)(19).
In addition to its unique enzymatic properties, the physical properties of gp1.7 are also intriguing. Its salt-precipitating property proved an asset in protein purification by serially salting in/out (2). The insolubility of the protein in the presence of salt is puzzling as its activity is not affected by the presence of NaCl or KCl up to 500 mM. One interpretation is that the salt promotes association of gp1.7 molecules to form a large matrix but does not affect the functional conformation of the enzyme. Indeed, gp1.7 is found as a large complex in solution even in the absence of salt. We estimate by MALS analysis that these large complexes each contain 12 monomers of gp1.7. This oligomerization could explain the low solubility of gp1.7 if, in the presence of salt, larger complexes lead to aggregation. The oligomerization of gp1.7 into dodecamers also explains why the two forms of gp1.7 co-purify by Ni-NTA chromatography even when only the large form has the His tag attached.
In light of the fact that all other known nucleotide kinases have a strict requirement for a divalent cation for activity, we wanted to examine for the presence of a sequestered metal ion. An analysis of the metal content of the purified enzyme showed that only Zn 2ϩ was present at a small, but significant level, in the 22-kDa and the wild-type species. Full-length gp1.7 (22-kDa) has 9 cysteine residues arranged in two putative zinc binding motifs: 21 CX 2 CX 10 C 35 and a 67 CX 3 CX 4 CX 2 C 79 (21) within the N-terminal half. Results suggest that at least the first three cysteines are critical for binding to zinc, as the 18-kDa gp1.7 lacking the N-terminal 41 amino acid residues binds considerably less zinc. The first 41 amino acids of gp1.7, however, are not required for conferring sensitivity of phage T7 to ddT in vivo (2) or for kinase activity of the purified 18-kDa protein in vitro (Fig. 1B). Because there are no other metal ions present at a significant level, we conclude that no divalent cation is required for the kinase activity of gp1.7. This finding is interesting because all other known enzymes that catalyze phosphate transfer have an absolute requirement for either a loosely bound (22) or a tightly bound divalent cation (16).
Interestingly, despite not requiring Mg 2ϩ for activity, the presence of Mg 2ϩ does have either a stimulatory or inhibitory effect depending on the acceptor/donor combination. In general, the presence of Mg 2ϩ stimulates activity when the acceptor/donor combination contains either dTMP or dGTP and inhibits activity when the acceptor is dGMP in combination with donor dTTP, dITP, ribo-GTP, or dUTP. Because Mg 2ϩ complexes directly with the phosphate groups of the nucleotide substrates, these complexes likely interact with the enzyme differently than the nucleotides without any bound metal ions.
Both dTMP and dGMP kinase reactions are reversible. We have shown that there are distinct regulatory controls toward thymidine and deoxyguanosine substrates by gp1.7. dGDP is a competitive inhibitor of both dGMP and dGTP. The results suggest that nucleotide binding sites for dGDP are identical or overlap with that of dGTP and dGMP, respectively. The K eq of 0.85 for dGMP obtained with gp1.7 is similar to that found for AMP in the E. coli adenylate kinase reaction. E. coli adenylate kinase catalyzes the conversion of AMP to ADP with ATP as donor (24). X-ray crystallography and NMR studies of E. coli adenylate kinase also revealed two nucleotide binding sites, one for ATP or ADP and the other for AMP or ADP (23,24). In contrast, the K eq of 0.07 for dTMP obtained with gp1.7 is much lower than that for dGMP. This finding suggests that the product dTDP gives strong inhibition and that a high concentration of dTTP is required to produce dTDP. Inhibition studies, however, show that dTDP is only a weak inhibitor of dTMP. Lineweaver-Burk plot analysis indicates a mixed type of inhibition by dTDP. These results taken together could be interpreted to mean that the enzyme complexes have separate binding sites for dTTP and dTDP as donor. This interpretation is supported by the observation that dTDP has no inhibitory effect on dGTP as donor (data not shown).
We hypothesize that binding of dTDP to the enzyme (dTDPsite) prevents the dissociation of the products dTDP from the enzyme complex, thus interfering with the binding of both dTTP and dTMP as donor and acceptor, respectively. Therefore, to shift the equilibrium of dTMP kinase reaction in a forward direction in the presence of dTDP, dTTP has to serve not only as a donor but a competitor with dTDP for the dTDP site, i.e. more dTTP is required. Consequently, binding of dTTP to the dTDP site would prevent the binding of dTDP, but allows dTMP to bind to acceptor site of the enzyme. In support of this hypothesis, dTTP and dTMP are present 2-fold higher than dTDP at equilibrium in a dTMP kinase reaction that contained equivalent amount of dTTP and dTMP at the outset ( Table 4). The apparent K m obtained with dTTP for dTMP kinase is 3-fold higher than that obtained for dGTP in the dGMP kinase reaction, indicating that an unusual high concentration of dTTP is required to saturate the enzyme. Additionally, the model can also explain why only a small amount of [ 3 H]dTTP can be produced at equilibrium in a reaction containing excess dTTP. gp1.7, however, does not display a clear sigmoidal dependence on low concentrations of dTTP in the dTMP kinase reaction, a property typical for homotropic allosteric modulators with positive cooperative binding (25). One possible explanation is that dTTP acts as activator of dTMP kinase only when dTDP is present.
These studies suggest that the mechanism of gp1.7 might likewise be quite complex in vivo. However, in T7-infected cells, synthesis of DNA occurs at a rapid rate, perhaps 5-10-fold preinfection (4,5). Therefore, the nucleoside triphosphates like dTTP and dGTP are unlikely to accumulate. The kinase reaction of gp1.7, therefore, would favor the production of dTDP and dGDP, which would be rapidly converted to the corresponding nucleoside triphosphates by the highly active host nucleoside diphosphate kinase (26) (EC 2.7.4.6). We previously showed that dTMP is exponentially converted to dTTP by the joint action of T7 gp1.7 and E. coli nucleoside diphosphate kinase using ATP as the ultimate phosphate donor (2). Note that in addition to being a precursor for DNA synthesis, dTTP is also used by the T7 gene 4 helicase to provide energy for the unwinding of DNA (27). The product of dTTP hydrolysis in the helicase reaction is dTDP. The amount of dTDP formed by helicase function is considerable as the hyrolysis of dTTP is required for the unwinding of just a few nucleotides. Thus, if the dTDP accumulated appreciably, perhaps by compartmentalization, then it could be recycled by gp1.7 as dTTP to the helicase. Furthermore, dGTP is a substrate of E. coli dGTPase, an enzyme that degrades dGTP to guanosine and tripolyphosphate (28). Thus one function of gp1.7 may be to maintain the appropriate balance of dNTPs that is critical for DNA replication fidelity (29). Perhaps the delay in the onset of maximal DNA synthesis in the absence of gp1.7 (1) reflects the an imbalance of the nucleoside triphosphate pool.
Finally, it is puzzling that gp1.7 does not share any sequence homology to other known nucleotide kinases. This lack of homology suggests that gp1.7 functions by a novel mechanism. It is tempting to speculate that the dodecamer plays a functional role in the metal-independent reaction, perhaps forming a protein based compartment (30) in which the critical residues from monomer are either components of the catalytic interfaces or are involved in subunit assembly. We have shown that even a single amino acid alteration in the C-terminal half of gp1.7 renders T7 phage resistant to ddT (1). The crystal structure of this remarkable enzyme should provide insight into the molecular basis of its catalytic activity.