Kinetic and Photochemical Studies of 3-N-Methyl-5-iodo-2’-deoxyuridine SENSITIZATION OF ULTRAVIOLET INACTIVATION OF THYMIDIXE I<Ir\‘ASE BY 3-N-METHYL-5-IODO-2’-DEOXYURIDIXE An-D OTHER HALOGENATED ANALOGS OF THYMIDINE*

The effects and a new analog of thymidine, on the ultraviolet (UV) inactivation of thymidine kinase been investigated. Of these compounds only the rate of inactivation of the enzyme The other

The other halogenated analogs neither protect nor sensitize thymidine kinase to W inactivation. Whereas the inactivation of thymidine kinase by W light in the presence of 5-iodo-2'-deoxyuridine can be prevented by the substrate, thymidine ( Kinetic studies with thymidine kinase show 5-iodo-2'-deoxyuridine to be a competitive inhibitor with respect to thymidine; however, 3-N-methyl-5-iodo-2'-deoxyuridine shows uncompetitive inhibition with thymidine and competitive inhibition with MgATP.
The primary photostable compound formed during photolysis of 3-N-methyl-5-iodo-2'deoxyuridine appears to be 3-N-methyl deoxyuridine, which is analogous to the formation of deoxyuridine during photolysis of S-iodo-2'-deoxyuridine ( in bacteria by Greer and Zamenhof (1) and by Greer (2) and in mammalian cells by Djordjevic and Szybalski (3). The photosensitivity of the various halogenated pyrimidine and deoxyribonucleoside derivatives is dependent upon many factors which include pH, frozen or nonfrozen state, the presence of organic substances, and the prcscncc or absence of oxygen (4 -6). Thus bromouracil and 5-bromo-2'.deoxyuridine were unaffected when irradiated in water or in the frozen state (7-9), but when irradiated in the presence of other pyrimidine derivatives or at high pH a marked phot'olability of bromouracil was observed (9, 10).
Bromouracil or iodouracil, when incorporated into DKh, not only is in a highly structured environment but also is in intimate proximity to other organic components of DNA. Hence, as observed, 5-bromouracil in DNA is very sensitive to photochemical alteration, with uracil being a major photoproduct (8). Rupp and Prusoff (5) observed that UV irradiation of 5-iodouracil results in the formation of a uracilyl free radical which undergoes a number of subsequent reactions dependent upon the composition of the reaction mixture during irradiation (5,6). Thus, irradiation of iodouracil in the presence of a substance from which a hydrogen can be abstracted by the uracilyl free radical results in the formation of uracil (5). The mechanism for the formation of DNA-uracil from DNA-bromouracil is similar to that observed during photolysis of 5-iodouracil in solution (8,(11)(12)(13). Recent studies of the photochemistry of 5-bromouracil(14) and of 5-fluorouracil (15)(16)(17) confirm that dehalogenation results from photolysis, however, the primary photoproduct of fluorouracil and its nucleosides is the hydrate which is subsequently dehalogenated to barbituric acid by elimination of HF (15). The photochemistry of 5-chlorouracil has not been as thoroughly studied as that of 5-fluoro-, 5-bromo-, or 5-iodouracil.
However, in view of the bond-dissociation energy of the carbon-chlorine bond (66. oro-and 5-iodo-2'-deoxyuridine were appreciably photoreactive as measured by a decrease in absorbance (4).
Elkind and Whitmore (19) commented that although it is reasonable to associate increased sensitivity of cells to irradiation with the incorporation of 5-chloro-, 5-bromo-, and 5-iodo-2'deosyuridine with cellular DNA, unequivocal evidence has not been presented that the DNA4 is the principal or sole target molecule.
The various other biochemical sites of inhibition exerted by 5-iodo-2'-deoxyuridine and its phosphorylated derivatives have been reviewed (20).
Recent investigations by Cysyk (21) have demonstrated that 5-iodo-2'-deoxyuridine, when present at the active site of thymidine kinase, markedly augments the rate of inactivation of the enzyme by GV light (253.7 am), and that thymidine not only decreases the rate of UV inactivation but also prevents the sensitizing effect of 5-iodo-2'-deoxyuridine.
Spectral Analyses-UV spectral analyses were conducted in a Cary model 15 spectrophotometer.
The mixture was allowed to stand overnight at room temperature.  All irradiation doses were corrected for the absorbance of the material being irradiated as described by Morowitz (24).

Fig. 2 compares the effects of several halogenated
nucleosides on the UV inactivation of thymidine kinase, and all appear to follow first order reaction kinetics. IdUrd and N-MeIdUrd enhance the UV inactivation of the enzyme, whereas 5-bromo-, 5-chloro-, and 5-fluoro-2'-deoxyuridine have little or no effect on either protecting or increasing the rate of inactivation. At equal concentrations of the nucleoside analogs, the sensitization of the enzyme to UV light is greater for IdUrd as compared with N-MeIdUrd.
However, unlike IdUrd, N-MeIdUrd is not phosphorylated by thymidine kinase but acts as an inhibitor of the enzyme.
Figs. 3 and 4 show that two different types of inhibition are produced by IdUrd and N-MeIdUrd when deoxythymidine is the variable substrate.
Both compounds bind to thymidine kinase, but their sites of binding are obviously different. As expected (Fig. 3  with thymidine ( Fig. 4) but competitive inhibition with MgATP (Fig. 5). The K; values were calculated from these inhibition patterns to be 20 PM for IdUrd and 1.7 mM for N-MeIdUrd, an indication that IdUrd binds more strongly to the enzyme. Cysyk (21) has demonstrated that when thymidine kinase is irradiated in the presence of IdUrd the increased rate of inactivation is specific and directed to the active site of thymidine.
The effect of increasing concentrations of N-MeIdUrd on the rate of UV inactivation of thymidine kinase is shown in Fig. 6. The concentration of N-MeIdUrd at the half-maximal rate of inactivation was calculated to be 1.4 mM, which is the same order of magnitude as the K; calculated from the kinetic inhibition studies (Figs. 4 and 5). Therefore, the sensitization caused by  N-MeIdUrd, like IdUrd (al), appears to be specific and directly related to the binding of the compound to thymidine kinase. Furthermore, Fig. 7 shows that the increased rates of inactivation of the enzyme caused by N-MeIdUrd and IdUrd arc independent of each other and are additive.
Since Cysyk (21) has shown that thymidine can prevent the enhancement of UV inactivation by IdUrd the effect of the substrates thymidine and MgATP on the sensitization of thymidine kinase by N-MeIdUrd was investigated. Neither thymidine nor MgATP alone decreased the sensitization by N-MeIdUrd, but a combinalion of both substrates (thymidine + MgATP) prevented almost completely the inactivation caused by N-MeIdUrd (Table I).
It should be noted further that a combination of MgATP and thymidine per se does not protect significantly against inactivation by UV light. Iwatsuki and Okazaki (25) have shown that the nucleotides, dTTP and dCDP, cause dimerization of thymidine kinase from Escherichia coli.
The former is an allosteric inhibitor, and the latter is an allosteric A comparison of the UV spectra of IdUrd and N-MeIdUrd before and after irradiation at 253.7 nm (Fig. 9A) shows that both halogenated derivatives undergo a decrease in their maximum at 285 nm and a hypochromic shift, which is indicative of dehalogenation.
After irradiation the mixtures were heated in acid which resulted in predominant peaks at 264 nm (Fig. 9B). This wave length is close to the optimum of 262 nm for deoxyuridine as well as for 3-N-MedUrd (27).
The subsequent dehalogenation of the hydrated fluorouracil derivative does not involve a free radical mechanism but rather an elimination reaction. This accounts for the apparent paradox of 5-fluoro-2'-deoxyuridine having a greater photolability than IdUrd yet not sensitizing thymidine kinase. The primary photochemical product is the hydrate when solutions of 5-fluorouracil, 5-fluoro-2'-deoxyuridine, N-methyl-5fluoro-2'-deoxyuridine or 5-fluorouridine-5'.phosphate are irradiated with UV (15). Spectral analyses of the irradiated solutions of IdUrd and N-MeIdUrd in 0.05 M Tris buffer at pH 7.8 ( Fig. 9) indicate that dehalogenation occurs with the formation of a substance that has a maximum absorbance at lower wave lengths.
Studies of the photochemical transformation of IdUrd by Cysyk (21) showed unequivocally that dUrd is the initial stable photoproduct formed, and, in analogy to the photochemistry of 5-iodouracil (5, 6), dUrd is formed via formation of the deoxyuridine free radical with subsequent abstraction of a hydrogen from the Tris buffer. Upon continued irradiation, however, deoxyuridinc forms a hydrate which can be reverted back to deoxyuridine by heating in acid (21). When the irradiated solution of N-MeIdUrd was acidified and heated (Fig. 9B), an increase in absorbance with a peak of 264 nm was observed.
These findings are in accord with the formation of a deoxyuridine hydrate analogous to that observed with IdUrd ( Fig. 9B (21)).
Since the spectral studies of IdUrd and N-MeIdUrd indicate a similar behavior, one may assume that a similar photochemical reaction mechanism is involved. Studies by Cysyk and Prusoff (26) and Cysyk (21) on the enhanced UV inactivatioil of thymidine kinase by IdUrd have shown that the sensitizing effect. of the halogenated nucleoside is active site directed and related to its binding constant.
The present study shows that although both N-MeIdUrd and IdUrd increase the rate of inactivation of thymidine kinase, N-MeIdUrd is not a substrate at the thymidine binding site, but rather iuhibits the enzyme uncompetitively with respect to thymidine and competitively with MgATP (Figs, 4 and 5). Like IdUrd (21), the rate of inactivation caused by N-MeIdUrd is related to the binding potential of the compound to the enzyme (Fig. 6). The K, values calculated from the kinetic plots indicate that the binding of IdUrd to thymidine kinase is approximately loo-fold greater than N-MeIdUrd and because of this one might expect proportionately a greater IJV inactivation with IdUrd as compared with the methyl derivative. However, this does not seem to be the case since at equal concentrations (Fig. 2) the inactivation caused by IdUrd is only twice that of N-MeIdUrd. Since N-MeIdUrd, (a) unlike IdUrd, is not phosphorylated by the enzyme, (b) has different inhibition patterns than IdUrd, and (c) appears to inactivate the enzyme by UV independently from IdUrd (Fig. 7), it is most probable that the site of binding of N-MeIdUrd to thymidine kinase is not only different than IdUrd but also more susceptible to UV  Product inhibition studies carried out by Voytek and Prusoff2 have revealed that the reaction mechanism of thymidine kinaee from E. co& is Iso Ordered Bi Bi with thymidine binding to the enzyme before the second substrate, MgATP.
UV inactivation experiments indicate that neither MgATP nor thymidine alone can overcome the enhanced inactivation caused by N-MeIdUrd (Table I) ; however, a combination of both substrates (thymidine + MgATP) does exert marked protection. This is in agreement with our study of the reaction mechanism of this enzyme, which indicated that thymidine must be bound to the enzyme prior to the addition of MgATP of which N-MeIdUrd is a competitive inhibitor.