Multisubstrate Analogs for Deoxynucleoside Kinases

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY 1985 by The Amerian Soiety of Biologial Chemists, n. Vol. 26, No. 23, ssue of Otober 15, pp ,1985 hinted in USA. Multisubstrate Analogs for Deoxynuleoside Kinases USE N NOVEL AFFNTY MEDA APPLED TO RESOLUTON OF LACTOBACLLUS ENZYMES* (Reeived for publiation, April 11, 1985) Seiihiro keda and David H. vess From the Department of Biohemistry, The Ohio State Uniuersity, Columbus, Ohio 4321 New multisubstrate-type inhibitors of the deoxynu- paired ativities do not share a ommon ative site in either leoside kinases have been synthesized, tested for their ase, a onvining onfirmation of bifuntional harater speifiity as soluble inhibitors of enzymes from La- would be the o-purifiation of two ativities on an affinity tobaillus aidophilus, and used to onstrut media for hromatography medium direted speifially to only one of affinity hromatography. Eah inhibitor was a deox- the ative sites. ynuleoside 5 adenosine 5 -P1,P4-tetraphosphate Although deoxynuleoside kinases, in general, are highly (abbreviated dnp4a, where dn represents a dado, speifi toward their deoxynuleoside substrates, only limited dcyd,dguo,ordthd moiety linked through its 5 - progress has been made in developing generally appliable hydroxyl to the terminal phosphate of adenosine tetraaffinity hromatography systems for this lass of enzymes by phosphate). At miromolar onentrations, eah inhibitor strongly and speifially inhibited the orrespondusing derivatized deoxynuleosides. The most useful media, ing deoxynuleoside kinase. Eah of the four Latobaillus deoxynuleoside kinase ativities was seletively retained on its homologous dnp4a-sepharose affinity medium. The ativity was eluted on addition of the respetive dnp4a with to purify thymidine kinase from several soures (5, 6). The up to 7% reoveryand 3-5-fold purifiation analogous media were reently prepared in our laboratory (relative to an ammonium sulfate fration). Whereas dthd kinase was retained only by the dtp4a olumn, a portion of the dado kinase ativity was retained, along with all the dcyd kinase ordguo kinase, on dcp4a- or dgp4a-sepharose, respetively, and oeluted with these ativities. Conversely, all three ativities were quantitatively retainedondap4a-sepharose, without ompetition from either dcyd or dguo, and were eluted simultaneously upon addition of dap4a. These observations further onfirm the under- standing that this organism employs paired, and presumably bifuntional, kinases, namely dcyd/dado kinaseanddguo/dado kinase, along with aseparate thymidine kinase. Reent work in this laboratory has shown that Latobaillus aidophilus R-26 has an unique multifuntional enzyme, dguo/dado kinase, in whih distint sites for dguo and dado phosphorylation reside on a single polypeptide hain (1). These ative sites displayed positive allosteri interations and ould be differentially inativated by hemial modifiation. n earlier reports (2, 3), we desribed paired ativities from the same strain direted toward dcyd and dado phosphorylation. Although the dcyd/dado kinase was similar to dguo/dado kinase in many respets, its bifuntionality ould not be proven due to the strutural lability of the protein. Sine it is lear, from several lines of evidene, that these * This work was supported in part by Grants PCM-8233 and DMB from The National Siene Foundation. A preliminary aount of this work was presented at a meeting of The Amerian Soiety of Biologial Chemists (ves, D. H., keda, S., and Chakravarty, R. (1984) Fed. Pro. 43,171). The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked aduertisemnt in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. : To whom reprint requests should be sent so far, have been those based on derivatization of the 3 - hydroxyl group. Thymidine-3 -(4-aminophenylphosphate) was oupled to Sepharose through a arboxy-terminal linker group by Kowal and Markus (4). This medium has been used from the 3 derivatives of ado, dcyd, and dguo and tested with a variety of deoxynuleoside metabolizing enzymes (7). Eah of these media performed very well with at least one enzyme, inluding beef liver mitohondrial dguo kinase and dcyd/dthd kinase and barley nuleoside phosphotransferase. The o-purifiation of mitohondrial dcyd and dthd kinase ativities on dcyd-sepharose onfirms experiments by Cheng et al. (5,6), who found that these ativities were retained and eluted together from dthd-sepharose. These parallel experiments on exatly analogous affinity media provide very onvining evidene that a single mammalian mitohondrial enzyme phosphorylates both dcyd and dthd. The above useful appliations of 3 -deoxynuleoside-sepharose olumns notwithstanding, in many instanes the attahment of a linker group to a deoxynuleoside drastially weakened its binding by an enzyme. As a onsequene, a number of enzymes, inluding the deoxynuleoside kinases from Latobaillus aidophilus, were not retained by these olumns (7). This observation, along with a quantity of unpublished work involving derivatization of purine or pyrimidine groups, has onvined us that the inherent nuleoside speifiity of the deoxynuleoside kinases makes most of them intolerant of linker groups attahed to the base or sugar of the deoxyriboside. On the other hand, ATP or other nuleoside triphosphates would be expeted to be a relatively nonspeifi affinity ligand. Sine nearly all nuleoside kinase reations proeed through a ternary omplex of enzyme, ATP, and nuleoside, we wondered if linking these substrates into multisubstrate analogs would reate more tightly bound affinity ligands whih might be linked to Sepharose through the relatively nonspeifi ATP end of the moleule. n an analogous ase, Lienhard and Seemski (8) disovered that Pl, P5- di(adenosine-5 )-pentaphosphate is a very potent inhibitor of rabbit musle adenylate kinase. ApSA has one more phosphate group than the linear ombination of the two substrates, AMP and ATP, and is far more inhibitory than the tetraphosphate

2 1266 dnp& Bisubstrate Affinity Media for Deoxynuleoside Kinases homolog, presumably beause it more nearly approximates the geometry of the transition state struture. Coupled to Sepharose, the pentaphosphate ompound effetively retained adenylate kinase (9). n the present study we have synthesized multisubstrate analogs for the deoxynuleoside kinases and have demonstrated their utility as ligands for affinity hromatography. EXPERMENTAL PROCEDURES Materials-Ap2A, ApSA, ApA, ApeA, and Ap4 were from Sigma. Tritiated deoxynuleosides were supplied by CN and Amersham Corp. Carbonyldiimidazole was from Aldrih. Diphenyl phosphorohloridate, sodium n-periodate, and adipi aid dihydrazide-sepharose were obtained from Sigma. Dimethyl formamide, dioxane, pyridine, and hexamethylphosphortriamide used for hemial syntheses were distilled over alium hydride and stored over 4-A moleular sieves from Union Carbide Cop. Diethyl ether was dried over moleular sieves. Bradford dye reagent for protein assay was from Bio-Rad and fluoresamine was from Rohe. Snake venom phosphodiesterase, nuleotide pyrophosphatase from Crotalus atroz venom, and alkaline phosphate from E. oli were obtained from Sigma. Polyethyleneimineellulose plates for TLC (Polygram CEL 3 PE/UV254) were from Maherey and Nagel Co. Enzyme Preparation and Assays-The ammonium sulfate fration (Fration V) ontaining unresolved dthd, dcyd, dguo, and &do kinases was prepared from Latobaillus aidophilus R-26 (ATCC 1156) as desribed previously (2, O). This fration was used for most of the experiments on inhibition and affinity hromatography. The deoxynuleoside kinase assays were arried out radiometrially (2, 1). The standard assay mixture ontained,l M Tris-HC1 (ph 8., 2 "), 1 mm ATP, 12 mm MgC,,.2 mm deoxynuleoside, and.5 pci of [3H]deoxynuleoside in.8 ml. From previous work, it is known that the dado kinase omponent of dcyd/dado kinase and dguo/dado kinase is stimulated up to 5-fold and 3-fold, respetively, by addition of dcyd and dguo (1, 3). Therefore, 2 mm dcyd and dguo were added to the above reation mixture whendado kinase was to be assayed in the fully ativated state. For experiments involving inhibition by dnpa,' redued substrate onentrations were used ATP,.3 mm; MgC12,.36 mm; and deoxynuleoside,.1 mm. n ontrast to dcyd, dguo, and dado kinases, whih show normal hyperboli ATP saturation urves, dthd kinase was found to follow a sigmoidal saturation urve with ATP. Therefore, nearly saturating ATP (5 mm) and MgClz (6 mm) were used in its assay. n the presene of 1 mm dctp, dthd kinase exhibited hyperboli kinetis with variable ATP. Therefore, dnpa inhibition of dthd kinase was studied with 1. mm dctp,.77 mm ATP, and 2.12 mm MgC, in the above assay mixture. Protein determinations were performed by the Bradford (11) or fluoresamine (12) methods. Preparation of dnp& Derivatives-Eah dnp4a was synthesized by oupling an adenosine polyphosphate omponent (ADP, ATP, or Ap4) to the deoxynuleoside monophosphate omponent previously ativated with arbonyldiimidazole or with diphenyl phosphorohloridate. The typial oupling reation was arried out in one of two ways. n Method A, arbonyldiimidazole was used as a oupling reagent, aording to the method of Bornemann and Shlimme (13), who prepared several (d)np,(d)n derivatives. ATP (.6 mmol) was reated with.2 mmol of damp previously ativated with arbonyldiimidazole. After the reation, the residue was dissolved in 5 ml of methanol/water (1:l) and applied to a olumn (3 X 15 m) of DEAE- Sephadex A-25 in the biarbonate form. After washing with 6 ml of methanol/water (M), a -.6 M linear gradient (2 X 1) of triethylammonium biarbonate (ph 7.5) was applied. The dap4a peak emerged from the olumn after ATP, but before the Ap4 by-produt was olleted. After evaporation of water, the residual triethylammonium biarbonate was removed by repeated evaporation from methanol. The yield of dap& was 16% relative to the damp starting material. Similar proedures were employed for preparation of dgpa (from dgmp and ATP). 259 ' The abbreviations used are: dnp4a, deoxynuleoside 5"adenosine 5"'-P',P'-tetraphosphate; ApnA, P1,P"-di(adenosine 5')-polyphos- 259 phate; (d)np,, (deoxy)nuleoside 5'-polyphosphate; Hepes, 4-(2-hydroxyethy1)--piperazineethanesulfoni aid. 261 n Method B, diphenyl phosphorohloridate was used as a oupling reagent. For reasons unknown, attempts to prepare dcp,a or dtp4a by Method A resulted in muh lower yields than were obtained with the purine derivatives. However, satisfatory yields of the pyrimidine analogs were obtained after ativation with diphenyl phosphorohloridate. The proedure used was essentially the method of Feldhaus et al. (91, who used it to prepare ApnA. ATP (.12 mmol) was reated with.2 mmol of dcmp previously ativated with diphenyl phosphorohloridate. The reation produts were separated as in Method A. dcpa eluted slightly after ATP. The ontaminating ATP was then removed by rehromatography on a olumn (3 X 15 m) of DEAE-Sephadex in the formate form. A.4-.7 M linear gradient (2 X lo ml) of ammonium formate buffer (ph 5.4) eluted a peak of pure dcp&. After evaporation of water, the ammonium formate was removed by sublimation at 65 "C. The yield of dcp4a (.18 mmol) was 16%, based on ATP. A similar syntheti proedure was used for the preparation of dtpa from dtmp and ATP. Charaterization of dnpj1"the purity of eah dnp4a was analyzed by high performane liquid anion exhange hromatography on a Pharmaia FPLC system equipped with a Mono-& olumn, using a linear -.5 M linear gradient of piperazine-hc1 (ph 6.), and by TLC on polyethyleneimine-ellulose plates, using.5 M triethylammonium biarbonate (ph 7.5). The order of elution from DEAE- Sephadex reflets the net harge on the nuleotide derivatives, judging from the behavior of known ompounds, inluding Ap2A, Ap3A, Ap&, and ApsA, along with the starting materials, dnmp and Apn. At ph 7.5, eah dnpa was eluted from DEAE-Sephadex between ATP and Ap4, refleting its four full negative harges. By ontrast, when the dnpa was eluted from the Mono-& anion exhange olumn at ph 6., it appeared after Ap4. Evidently, interation between the two base moieties of the mp4a and the matrix struture of the Mono-Q olumn results in delayed elution of dinuleotides ompared with mononuleotides of equal, or even greater, net harge. None of the dnpa ompounds was degraded by E. oli alkaline phosphatase, exluding the possibility of the presene of terminal phosphate ester groups. However, digestion with snake venom phosphodiesterase or nuleotide pyrophosphatase gave dnmp and AMP as final produts in eah ase. These results are onsistent with the struture predited for eah dnp.,a. The UV spetra of eah dnpa were taken at aidi, neutral, and basi phs (Table ). For omparison, the spetra of equimolar mixtures of eah pair of omponent nuleotides were examined. The spetrum of eah mixture was found to be the exat sum of the spetra of the individual mononuleotides, having a Amax at an intermediate wavelength, and no learly defined shoulders (not shown). The., X (Table ) and shape of spetrum observed for eah dnp4a was preisely the same as that of the equimolar mixture of the twoomponent nuleotides. This result supports the view that eah dnp,a we synthesized ontains the two-omponent nuleotides in a 1:l ratio. Upon omplete enzymati hydrolysis by nuleotide pyrophospha- tase, the absorbane of eah dnpd at its,, X inreased up to 13%. This hyperhromi phenomenon was most pronouned at ph 7.2, ompared with aidi or alkaline onditions. The overall shape of eah spetrum remained unhanged by hydrolysis. These results indiate that the two base portions in eah dnp4a are staked to some extent under most onditions, produing the hypohromi effets observed. The absorption oeffiient () for eah dnp& in Table TABLE r Ultraviolet spetral properties of dnpd The absorption spetra were obtained as desribed under "Experimental Proedures," using a Gilford spetrometer reently alibrated with wavelength and absorbane standards. The molar extintion oeffiients are the values in parentheses., X, (nm); e (M-' m" X 1").1 N HCl ph N NaOH dcp4a (19.4) (2.8) (21.1) dtpa (21.6) (23.5) (21.5) dap4a (26.7) (26.3) (26.7) dgpra (23.) (24.6) (23.)

3 dnpdp Bisubstrate Affinity Media for Deoxynuleoside Kinases was alulated from the spetrum of an equimolar mixture of the omponent nuleotides whose onentrations were standardized using published molar extintion oeffiients for eah omponent (14) and applying the appropriate hypohromi fator mentioned above. The total phosphate ontent of eah dnpd was determined (15) after total hydrolysis with aidified Mg(NO& The molar quantity of dnp4a was alulated from its absorbane at its, X, at ph 7.2, using the absorption oeffiient from Table. The following molar ratios of phosphate/dnp& were obtained dcpd, 3.61; dtp,a, 3.91; dap4a, 3.94; dgp,a, Further onfirmation of the struture for dcpa was derived from 'H NMR and 31P NMR. The 'H NMR spetrum (DzO, pd 7) was taken with a Varian T-6 NMR spetrometer and hemial shifts were measured relative to that for tetramethylammonium hloride. Several distint signals were able to be assigned by omparison with the spetra of the omponent nuleotides, dcmp and AMP (s, 1H for H-8 of adenine), 8.13 (s, 1H for H-2 of adenine), 7.73 (d, 1H for H-6 of ytosine), 2.35 (m, 2H for H-2' of deoxyribose). The resonanes for protons on the two base portions of dcp,a are shifted upfield by as muh as.2 As, ompared with those of the omponent nuleotides. These shifts may be interpreted as additional evidene for staking of the two bases in dnp4a (16). 31P NMR spetra were kindly run by Dr. M.-D. Tsai on a Bruker WP-2 NMR spetrometer, at 81.2 MHz. The sample ontained 1 mm dcp,a in.1 M K+/Hepes, ph 8., and 25% DzO. The hemial shifts were measured relative to 85% phosphori aid as a standard. Two resonane signals were observed, (d, 2P), (m, 2P). Although the fine struture has not been larified for either resonane, the hemial shift for eah signal is exatly omparable to that exhibited by the two a-phosphates and the respetively, of Apd (17), and the two a-phosphates and three middle phosphates, respetively, ofap5a (18). These results are totally onsistent with a 5', 5""tetraphosphate struture for dnp&. The metaboli stability of eah dnp4a toward enzymes in L. aidophilus was examined by inubating 1 mm final onentration of the ompound with about 1 times the protein onentration (Fration V enzyme) required for a normal kinase assay. After 3 min of inubation at 2 "C, no detetable breakdown was observed by polyethyleneimine-ellulose TLC. When this inubation was prolonged (24 h), minor spots of AMP and dnmp were seen in the enzymetreated samples, but not in samples inubated without enzyme. Although this result indiates that dnpd is suseptible to hydrolyti ativity found in the baterial extrat, no signifiant turnover or degradation of dnpd appears to our under assay onditions. Aqueous solutions of the dnp4as are quite stable upon storage at -2"C. After 3 months of storage, no appreiable breakdown was detetable by TLC. Preparation of Affinity Adsorbents-Eah dnpd was oupled to Sepharose by a method used previously for ATP-Sepharose (19). dnpd (5 pmol in 2 ml of water) was mixed with equimolar Na4, then inubated in the dark for 1 h at 4 "C. After the remaining periodate was quenhed with glyerol (18 pl of 1% aqueous solution), 2 ml of.4 M sodium aetate buffer (ph 5.) was added. One ml of adipi aid hydrazide-sepharose was suspeted in the oxidized dnpd solution. After gentle shaking for 24 h at 4 "C, the gel was thoroughly washed with water and 1 M NaCl. The amount of dnpa bound was estimated by determining the disappearane of oxidized dnp,a from the supernatant solution of the suspension. About 1. pmol of dnp& was bound per ml of gel in eah ase. The dtp4a-, dcpd-, and dgpd-sepharose produts were used for affinity hromatography without further modifiation of the remaining hydrazide residues. The dapd-sepharose was suspended in 5 ml of water at 4 "C and 5 pl of aetaldehyde were added. After shaking for 1 h at room temperature, the gel was thoroughly washed with water and 1 M NaC1. After this treatment, a olor test with 2,4,6-trinitrobenzene sulfoni aid (2) showed the disappearane of the remaining hydrazide groups. NO signifiant loss of apaity as an affinity adsorbent was observed after 1 year and about 1 yles of use. RESULTS nhibitory Speifiity of dnpa-the inhibitory effets of eah mp.& on deoxynuleoside kinases from L. aidophilus R26 was examined, using a Fration V (1) ammonium sulfate fration ontaining all four ativities. So that ompetitive effets ould be seen learly, subsaturating onentrations of deoxynuleoside (1 PM) and ATP (3 PM) were used in the assays. The mixed dado kinase ativities (from dcyd/dado and dguo/dado kinases) were assayed in the presene of their allosteri ativators, dcyd and dguo. Similarly, dthd kinase was ativated by dctp. By having these ativities in their fully ativated states, omplexities due to possible ativation by the dnp& are eliminated and the effets we observe presumably are due to binding of a dnpa to ative site of a deoxynuleoside kinase. As shown in Table 11, eah dnpra potently and speifially inhibited its orresponding deoxynuleoside kinase ativity. These results learly indiate that eah dnp,a is direted speifially toward the homologous kinase ative site but does not readily bind to any other kinase ative site. A orollary impliation is that eah deoxynuleoside is phosphorylated at a distint ative site. At onentrations at 5 pm, or more, some degree of rossinhibition by eah dnp,a was observed. Sine eah dnp4a has the features of an ATP analog at one end, it may, if suffiiently onentrated, bind to the ATP subsite of any kinase. The other end of the moleule, whih an be viewed as a deoxynuleoside triphosphate, might also bind to the phosphate-donor subsite, sine many kinases an use deoxynuleoside triphosphates as well as ATP as phosphate donors. As a hek on whether any dnpd binds to regulatory sites, the effets of eah dnpd on dado and dthd k. lnases were examined in the absene of ativators. dap4a inhibited dado kinase to exatly the same extent in the presene, or the absene, of ativators. dtp4a, at 1 PM, exhibited no effet on dado kinase in either ase. Therefore, neither dap,a nor dtp4a bind to a regulatory site for dado kinase. On the other hand, dcp4a or dgp.&, whih showed no signifiant effets on fully ativated dado kinase (Table 11), were found to inrease dado kinase ativity up to 2-fold in the absene of other ativators. Evidently, these bisubstrate analogs do bind to sites whih regulate dado kinase. As was quoted above, we have previously found that the respetive deoxynuleoside substrates of dcyd kinase or dguo kinase, or their analogs, stimulated the dado kinase ativities paired with these enzymes (1, 3). There was substantial hemial and kineti evidene to support the notion that, in eah ase, the ativation of the &do kinase is aused by the binding of dcyd or TABLE 11 nhibition of deoxynuleoside kinases from L. aidophilus R-26 by dnpa For eah inhibitor, the uo of the Fration V deoxynuleoside kinase reation was measured in the presene and absene of the dnp4. For dcyd, dguo, and dado kinases,.1 mm deoxynuleoside and.3 mm MgATP were used (roughly K,,, substrate onentrations). n addition, ativators were inluded in the dado kinase reation mixture: 2. mm dcyd and dguo. For the dthd kinase assay,.2 mm dthd and.77 mm ATP were used, along with the ativator, 1. mm dctp, and 2.12 mm MgC12. nhibitor Conentration % inhibition of kinase ativity ~~ dcyd dguo &do dthd PM dcpd dgpd &PA dtp

4 12662 dnpa Bisubstrate Affinity Media for Deoxynuleoside Kinases dguo, or their derivatives, to the dcyd kinase or dguo kinase ative site found on the same protein as the dado kinase. Therefore, the ativation of dado kinase observed in the presene of dcp& or dgp,a is also likely to be mediated by the binding of these ompounds to the dcyd or dguo kinase ative sites. Eah is speifially direted to its struturally analogous ative site, on the one hand inhibiting ativity there and, on the other hand, stimulating the ativity of the ounterpart dado kinase ative site. The effet of eah dnp& on dthd kinase was also examined in the absene of its ativator, dctp. The ATP substrate, whih is bound in a highly ooperative manner in the absene of dctp, was kept at the half-maximal ativity onentration of 3 mm. Under these onditions, dtp4a inhibited dthd kinase about as it did in the presene of dctp, whereas dcp4a, dgp4a, and dap4a all ativated dthd kinase up to about 3%. t seems that the latter three ompounds all have some affinity for the regulatory site of dthd kinase, although they do not bind signifiantly to the ative site of this enzyme (Table 11). Affinity Chromatography of Deoxynuleoside Kinases on dtp+4-sepharose-the probable struture of the dnp&- Sepharose derivatives is represented in Fig. 1. The side-hain struture an be expeted to ontribute minimally to hydrophobi binding, but, of ourse, some nonspeifi ation exhange is probable on the multiple negative harges of the phosphates. n our early work with the Latobaillus enzymes, thymidine kinase was reognized as distint from the other three deoxynuleoside kinases, as it ould be separated easily from them on alium phosphate gel (21). This is onfirmed by its hromatographi behavior on dtp,a-sepharose, as shown in Fig. 24. More than 8% of the dcyd, dguo, and dado kinase ativities were reovered in the run-through fration, along with more than 98% of the rude proteins. However, all of the dthd kinase was retained, even after the olumn was washed with 1 M KCl. Upon addition of.25 mm dtp&, 32% of the dthd kinase ativity was estimated to be in the eluted fration, making appropriate orretion for the inhibition by dtp4a in the assay mixtures. A large inrease in the speifi y42 yh2 = YH YNH '///////// Sepharose FG. 1. Struture of dnp4a-sepharose. The symbol B on the deoxyribose refers to the bases, Ade, Cyt, Gua, or Thy. The exat struture of the linkage between the hydrazide linker hain and the periodate-oxidized adenosine moiety is not known o 3.8 o.6 2 k Y - sr ' a, u).2 Q A. d.tp,a-seph'arose B. dap,a-sepharose. dcp4a-sepharose la. D. dg~,a-sepharose jb Fration No..~ o 2. n.- 1. t; m L U \ ul E Y E.2.- a,.1 L n n 1. ; Y.5 FG. 2. Affinity hromatographyofdeoxynuleoside kinases from L. aidophilus on dnp4a-sepharose., dthd kinase; A, dcyd kinase;, dguo kinase; and, dado kinase. A, hromatography on dtp4a-sepharose-.8 ml of gel was equilibrated at 4 "C with 2mM Tris-HC1, ph 8., ontaining 2% glyerol. Diluted Fration V (4.3 mg of protein in.5 ml of buffer) was applied in one olumn volume and allowed to stand 3 min; flow was then resumed with equilibration buffer. At arrow a, 1 M KC1 in equilibration buffer was started and at b elution buffer onsisting of.25 mm dtp4a and 1 M KC1 in equilibration buffer was applied. Frations were 1. ml. B, hromatography on dap4a-sepharose-1.4-ml gel was equilibrated with 5 mm K+/phosphate, ph 6., ontaining 2% glyerol. After equilibration with sample (2.8 mg of protein in.5 ml of buffer), the olumn was washed with equilibration buffer. At arrow a, washing with 2 mm Tris-HC1, ph 8., ontaining 2% glyerol and.2 M KCl, was started. At b, elution buffer (.1 mm dap4a in 2 mm Tris- HCl, ph 8., ontaining 2% glyerol) replaed the washing buffer. Frations were 1. ml. C, hromatography on dcp,a-sepharose-1. ml of gel was equilibrated with 2 mm Tris-HC1, ph 8., ontaining 2% glyerol and.1 mm dgpda. After equilibration with Fration V sample (.7 mg of protein in.5 ml of buffer), the olumn was washed with the equilibration buffer, then at a the dgpj was removed from the buffer. At arrow 6, elution buffer (.1 mm dcpj in 2 mm Tris-HC1, ph 8., ontaining 2% glyerol) was added. Frations were 1. ml. D, hromatography on dgpd-sepharose-2. ml of gel was equilibrated with 2 mm Tris-HC1, ph 8., ontaining 2% glyerol and.1 mm dcyd. After Fration V enzyme (1.4 mg of protein in.5 ml of buffer) was applied, the olumn was washed with equilibration buffer. At a, washing buffer (.1 M KC1 in 2 mm Tris- HCl, ph 8., ontaining 2% glyerol) was started. At b, the washing buffer was swithed to another mixture onsisting of.5 mm dapa in 2 mm Tris-HC1, ph 8., ontaining 2% glyerol. Finally, at, elution buffer, in whih.5 mm dgpj replaed the dap4a, was ommened. Frations were 2. ml.

5 dnp& Bisubstrate Affinity Media ativity of the eluted enzyme is likely, as there was too little protein to measure by the sensitive fluoresamine method. No dcyd, dguo, or dado kinase ativity was detetable in the dthd kinase frations. The binding of dthd kinase to the olumn appears to be very tight, sine other elutrients, suh as the ombined substrates (.1 mm dthd and 1 mm ATP) failed to release any ativity. Affinity Chromatography on dap.,a-sepharose-when the Fration V enzyme fration was applied to dap,a-sepharose, only dthd kinase ran through the olumn (with quantitative reovery), along with 95% of the protein (Fig. 2B). Although- as was mentioned above-the dapj ligand showed some affinity for the dthd kinase regulatory site as an ativator, this affinity is evidently too weak to result in retention of this enzyme on the dap4a-sepharose olumn. After washing with.2 M KC1 to remove any nonspeifially bound protein, the dcyd, dguo, and dado kinase ativities were eluted simultaneously by.1 mm dap4a. The ombhation of.1 mm dado plus 1 mm ATP was found to be a suitable replaement for the bisubstrate elutrient. Reovery of all three kinase ativities was about 6% and the speifi ativity was inreased 35-fold by this purifiation step. The purity of this fration was heked by onentrating it (2 yl) to about 25 pl and subjeting 5-yl aliquots to sodium dodeyl sulfate-polyarylamide gel eletrophoresis. However, no protein bands were detetable by Coomassie blue staining. Silver staining produed several weakly staining bands, but no lear band ould be visualized at the position where the homogeneous dguo/dado kinase showed up in our previous work (1). Apparently, the quantity of enzyme protein obtained was too small and a saled-up preparation would be required for detetion by eletrophoresis. Furthermore, another washing step (with dilute solutions of ATP, for example) may be needed to eliminate impurities retained by the olumn. Sine the purpose of this paper is not neessarily to purify these enzymes to homogeneity, we did not arry this effort any further. f.1 mm dado was added to the sample and to the olumn equilibration buffer (5 mm K+/phosphate, ph 6.), all of the dcyd, dguo, and dado kinase ativities ran through the olumn (results not shown). On the other hand, addition of.1 mm dcyd plus.1 mm dguo to the sample and equilibration buffer did not alter the retention-elution profile shown in Fig. 2B. Clearly, these results indiate that the dap,a ligand is direted only toward dado kinase ative sites, but not the ative sites for dcyd and dguo kinases. Therefore, the retention of the dcyd and dguo kinase ativities on the dap,a-sepharose must be by virtue of their assoiation with dado kinase. Affinity Chromatography on dcp.,a-sepharose-fig. 2C shows all of the dguo kinase omponent of Fration V running through the dcp4a-sepharose olumn, aompanied by 3% of the total dado kinase ativity. The dthd kinase also ran through, but for the sake of larity, these results are not shown. No detetable dcyd kinase was found in these frations. Following washing with equilibration buffer, the dcyd kinase was eluted-with more than 5% reoveryupon addition of 1 PM dcpj. Assoiated with this fration was 24% of the total dado kinase. n early experiments, about 5% of the dguo kinase was found in these frations, but the addition of 1 PM dgp,a to the sample and equilibration buffer ompletely eliminated this slight nonspeifi retention of dguo kinase. One again, these results onfirm that dado kinase ativity is assoiated with dcyd kinase. Affinity Chromatography on dgpd-sepharose-appliation of Fration V to dgp,a-sepharose (Fig. 2) resulted in for Deoxynuleoside Kinases retention of nearly all of the dguo kinase ativity, whereas dcyd kinase ran through, aompanied by 3% of the original dado kinase and 92% of the rude protein. dthd kinase also ran through this olumn (not shown). After washing with.2 M KCl, the dguo kinase ativity was eluted-with 7% reov- ery-upon the addition of 5 ~ L M dgp4a. This dguo kinase fration had assoiated with it 22% of the original dado kinase ativity. A small perentage of the dcyd kinase was also found under these frations in initial experiments, but addition of.1 mm dcyd to sample and equilibration buffer eliminated this ross-ontamination ompletely. When another elutrient (.5 mm dap,a in 2 mm Tris-HC1, ph 8., ontaining 2% glyerol) was added right before elution with dgpd, no signifiant dado kinase nor dguo kinase was eluted. These results indiate that dguo kinase is retained on the olumn by diret interation of its ative site with the dgpa ligand, whereas dado kinase was retained on the olumn by virtue of its assoiation with dguo kinase. DSCUSSON Taken together, the hromatographi data we have ited learly show that: (i) dthd, dcyd, and dguo kinases are separate proteins; (ii) both dcyd and dguo kinases have assoiated dado kinase ativities; and, (iii) on eah pair of kinase ativities, dcyd/dado kinase and dguo/dado kinase, there are distint ative sites for eah of its deoxynuleoside substrates. t has reently been established by kineti and strutural analysis that dguo and dado kinase ativities are funtions of distint sites on a single polypeptide hain (1). Our present observation that dgp,a speifially inhibits dguo kinase, but ativates dado kinase, is further kineti evidene for the multifuntionality of this protein. Moreover, by a totally different approah, the hromatographi patterns we have presented also onfirm that distint ative sites for dguo kinase and dado kinase reside on a single protein. Now, the possibility that we are dealing with very similar, but separate, kinases for dguo and dado an be exluded. Exatly parallel arguments seem to apply to the dcyd/dado kinase pair, as well. We do not know yet whether the dcyd and dado kinase ativities reside on a ommon polypeptide or if they are assoiated with separate but interating subunits. However, in their native ative states, the two pairs of ativities exhibited idential moleular weights (1) and eletrophoreti mobilities (2). Therefore, sine the dguo/dado ativities are found on the same monomeri protein, it seems likely that the dcyd/dado kinase ativities will also oupy a ommon polypeptide. However, that analysis does not lie within the sope of this paper. The hromatography on bisubstrate analogs in this study has aomplished what affinity hromatography with a variety of deoxynuleoside derivatives ould not: the seletive reten- tion, omplete separation, and elution of the Latobaillus deoxynuleoside kinases. Neither the 3'-linked deoxynuleoside olumns we reently desribed (7), nor amino-linked dado and dcyd and C8-linked dguo2 effetively retained any of these baterial enzymes. Evidently, the highly speifi binding of a deoxynuleoside by any of these kinases involves fairly rigid steri onstraints. On the other hand, as we have seen in the present study, the linkage of a deoxynuleoside through the 5' position, as part of a bisubstrate analog, does not seriously interfere with binding. The great speifiity of inhibition by eah dnp4a shown in Table 1 learly indiates * S. keda and D. H. ves, unpublished experiments.

6 12664 dnp& Bisubstrate Affinity Media that the deoxynuleoside portion of eah inhibitor binds speifially to the deoxynuleoside subsite of the orresponding kinase ative site. Whether the ATP portion of dnpa also binds to the subsite for ATP has to be determined. Details of the binding mehanisms for these multisubstrate analogs will be presented in a paper appearing elsewhere. n any ase, it seems lear that linking these analogs to Sepharose through the relatively nonspeifi and weakly binding ATP end of the moleule does not spoil the speifi affinities of these ompounds for kinases. We wish to suggest that, for enzymes whih form a ternary omplex with two substrates during the ourse of atalysis, onstrution of multisubstrate analogs may be the most reliable avenue to affinity hromatography. n spite of their potential usefulness, these powerful ligands have not been widely applied to affinity hromatography. One example, mentioned in the introdution to this paper, is the use of a diadenosine analog Apd-Sepharose for the removal of adenylate kinase (9). Another, developed by one of the authors, employs pyridoxyltryptophan in the isolation of tryptophanase (22). Multisubstrate.analogs should also be ideal vehiles for affinity labeling, as was suggested by Wolfenden s review (23). Na4-oxidized Ap,A has been used suessfully to loate the ApsA binding site of DNA polymerase (24) and derivatives of dnp,a may provide a onvenient means of mapping the ative sites of the deoxynuleoside kinases. REFERENCES 1. Chakravarty, R., keda, S., and ves, D. H. (1984) Biohemistry 23, Deibel, M. R., Jr., and ves, D. H. (1977) J. Biol. Chem. 252, Deibel, M. R., Jr., Reznik, R. B., and ves, D. H. (1977) J. Biol. Chem. 252, for Deoxynuleoside Kinases 4. Kowal, E. P., and Markus, G. (1976) Prep. Biohem. 6, Cheng, Y.-C., Domin, B., and Lee, L.-S. (1977) Biohim. Biophys. Ata 481, Lee, L.-S., and Cheng, Y.-C. (1976) J. Biol. Chem. 251, keda. S., Park... Gardner., P... and ves, D. H. (1984).. Biohemist& 23, Lienhard. G. E.. and Seemski ),, J. Biol. Chem Feldhaus, P., Frohlih, T., Goody, R. S., sakov, M., and Shirmer, R. H. (1975) Eur. J. Biohem. 57, Deibel, M. R., Jr., and ves, D. H. (1978) Methods Enzyml. 51, Bradford, M. M. (1976) Anal. Biohem. 72, Udenfreund, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigel, M. (1972) Siene 178, Bornemann, S., and Shlimme, E: (1981) 2. Naturforsh. 36, Dunn, D. B., and Hall, R. H. (1968) in Handbook of Biohemistry (Sober, H. A., ed) pp. G8-98, The Chemial Rubber Co., Cleve- land 15. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem. 66, Kolodny, N. H., Kisteneff, E., Redfield, C., and Rapaport, E. (1979) FEBS Lett. 17, Plateau, P., Mayaux, J.-F., and Blanquet, S. (1981) Biohemistry 2, Nageswara Rao, B.D., and Cohn, M. (1977) Pro. Natl. Aud. Si. U. S. A. 74, Lamed, R., Levin, Y., and Oplatka, A. (1973) Biohim. Biophys. Ata 35, Cuatreasas, P. (197) J. Biol. Chem. 245, Durham, J. P., and ves, D. H. (1977) Biohim. Biophys. Ata 228, keda, S., Hara, H., Sugimoto, S., and Fukui, S. (1975) FEBS Lett. 56, Wolfenden, R. (1977) Methods Enzyml. 46, Grummt, F., Waltl, G., Jantzen, H.-M., Hampreht, K., Huebsher, U., and Kuenzle, C. C. (1979) Pro. Nutl. Aud. Si. U. S. A. 76,