A QM-MD simulation approach to the analysis of FRET processes in (bio)molecular systems. A case study: complexes of E. coli purine nucleoside phosphorylase and its mutants with formycin A.

Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand - formycin A (FA). FRET occurs between an excited Tyr residue (D*) and FA (A). This study aims to interpret experimental data that, among others, suggests the absence of FRET for the PNPF159A mutant in complex with FA, based on novel theoretical methodology. MD simulations for the protein molecule containing D*, and complexed with A, are carried out. Interactions of D* with its molecular environment are accounted by including changes of the ESP charges in S1, compared to S0, and computed at the SCF-CI level. FRET probability W F depends on the inverse six-power of the D*-A distance, R da . The orientational factor 0 < k(2) < 4 between D* and A is computed and included in the analysis. Finally W F is time-averaged over the MD trajectories resulting in its mean value. The red-shift of the tyrosinate anion emission and thus lack of spectral overlap integral and thermal energy dissipation are the reasons for the FRET absence in the studied mutants at pH 7 and above. The presence of the tyrosinate anion results in a competitive energy dissipation channel and red-shifted emission, thus in consequence in the absence of FRET. These studies also indicate an important role of the phenyl ring of Phe159 for FRET in the wild-type PNP, which does not exist in the Ala159 mutant, and for the effective association of PNP with FA. In a more general context, our observations point out very interesting and biologically important properties of the tyrosine residue in its excited state, which may undergo spontaneous deprotonation in the biomolecular systems, resulting further in unexpected physical and/or biological phenomena. Until now, this observation has not been widely discussed in the literature.

Changing phenotypic expression in a patient with a mitochondrial encephalopathy due to 13042G>A de novo mutation--a 5 year follow up.

Mutations in NADH dehydrogenase (ND) subunits of complex I lead to mitochondrial encephalomyopathies associated with various phenotypes. This report aims to present the patient's clinical symptomatology in the context of a very rare 13042G>A de novo mutation and with an emphasis on changing phenotypic expression and pronounced, long-standing response to levetiracetam.

Idiopathic trigeminal sensory neuropathy. A case report.

Idiopathic trigeminal sensory neuropathy is a rare clinical condition characterized by sensory disturbances on the face. Its symptoms may be permanent or temporary and a wide variety of diagnostic procedures is usually required to establish the diagnosis. Frequently, it is the first manifestation of a systemic disorder. In the majority of cases causal treatment is not possible, even though patients with trigeminal sensory neuropathy should be carefully monitored by physicians.

Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine.

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kinetics, were applied to study the reaction of purine nucleoside phosphorylase (PNP) from Escherichia coli with its substrate N(7)-methylguanosine (m7Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (pK(a)=7.0), each with characteristic absorption and fluorescence spectra, over the pH range 6-9, where absorption and intrinsic fluorescence of the enzyme are virtually unchanged. The pH-dependence of kinetic constants for phosphorolysis of m7Guo were studied under condition where the population of the zwitterion varied from 10% to 100%. This demonstrated that, whereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than the cation for the mammalian enzymes, both ionic species are almost equally good substrates for E. coli PNP. The imidazole-ring-opened form of m7Guo is neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescence quenching, and concomitant changes in absorption and fluorescence spectra of the two ionic species of m7Guo on binding, showed that both forms are bound by the enzyme, the affinity of the zwitterion being 3-fold lower than that of the cation. Binding of m7Guo is bimodal, i.e., an increase in ligand concentration leads to a decrease in the association constant of the enzyme-ligand complex, typical for negative cooperativity of enzyme-ligand binding, with a Hill constant <1. This is in striking contrast to interaction of the enzyme with the parent Guo, for which the association constant is independent of concentration. The weakly fluorescent N(7)-methylguanine (m7Gua), the product of phosphorolysis of m7Guo, is a competitive non-substrate inhibitor of phosphorolysis (K(i)=8+/-2 microM) and exhibits negative cooperativity on binding to the enzyme at pH 6.9. Quenching of enzyme emission by the ligands is a static process, inasmuch as the mean excited-state lifetime, =2.7 ns, is unchanged in the presence of the ligands, and the constants K(SV) may therefore be considered as the association constants for the enzyme-ligand complexes. In the pH range 9.5-11 there is an instantaneous reversible decrease in PNP emission of approximately 15%, corresponding to one of the six tyrosine residues per subunit readily accessible to solvent, and OH- ions. Relevance of the overall results to the mechanism of phosphorolysis, and binding of substrates/inhibitors is discussed.

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer.

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.

Unusual nucleoside triphosphate donors for nucleoside kinases: 3'-deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate.

Two non-conventional analogues of ATP, 3'-deoxyadenosine-2'-triphosphate (3'-d-2'-ATP) and 2'-deoxyadenosine-3'-triphosphate (2'-d-3'-ATP), the syntheses of which are described, were examined as potential phosphate donors for the nucleoside kinases: 2'-deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2). The reactions were monitored by means of a mixture of [gamma-32P]ATP and cold analogue, and/or with the use of 3H-labelled acceptors and cold donor. With dCK, using equimolar mixtures of ATP with each analogue, and dC as acceptor, phosphate transfer from 3'-d-2'-ATP and 2'-d-3'-ATP amounted to 34% and 14%, respectively. With each analogue used alone (each at concentration of 100 microM), phosphate transfer from 3'-d-2'-ATP was 55% that from ATP, and from 2'-d-3'-ATP 16%. With human TK2, and equimolar mixtures of [gamma-32P]ATP with each of the analogues, and 1 microM dT as acceptor, there was no detectable transfer from either analogue. But, when each analogue was used alone, phosphate transfer attained 11% and 5%, respectively, that for ATP alone. With the low affinity form of human TK1, and dT as acceptor, only low phosphate transfer occurred with either analogue used alone. Both compounds exhibited Michaelis-Menten kinetics (with significantly lower Vmax than ATP), while ATP exhibited cooperative kinetics with all three kinases.

Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2).

Substrate/inhibitor specificities of nucleoside analogues with modified sugar moieties toward highly purified deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) from human leukemic spleen have been examined. Substrate activities of cytosine nucleosides vs dCK were as follows: 2'-fluoro-dC > 2'-O-methyl-C > araC > 2'-fluoro-2'-deoxy-araC > 3'-O-methyl-dC = 3'-fluoro-2',3'-ddC > cytosine beta-L-riboside > 2',3'-ddC > C = 1-(4-hydroxy-1,2,-butadienyl)-cytosine (cytalene) = 2'-azido-dC. Modified purine nucleosides were only feeble substrates: ara-A > 2'-fluoro-2',3'-dideoxy-araA = 2'-O-methyl-A. With TK1 and TK2, similar sugar-modified analogues of dU and dT were feeble substrates. Surprisingly alpha-dT was a relatively good substrate, as well some beta-L-ribonucleo-sides. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. The overall data are relevant to the role of these enzymes in "activation" (by phosporylation) of nucleoside analogues with antiviral and antitumor activities.

Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic N4-hydroxycytosine.

UV and NMR spectroscopy were employed to study the tautomerism, acid-base properties and conformation of the exocyclic N(4)-OH group in 1-methyl-N(4)-hydroxycytosine (1-mOH(4)C), and its methyl derivatives, viz. the fixed imino forms (1,3-m(2)OH(4)C and 1,3,5-m(3)OH(4)C), the fixed amino form (1,N(4)-m(2)OH(4)C), and analogues sterically constrained to the form syn (1,5-m(2)OH(4)C) or anti (1,3-m(2)OH(4)C) with respect to the ring N(3). Relative to 1,N(4)-m(2)OH(4)C, UV spectroscopy showed that the other analogues were predominantly imino and that all analogues formed a structurally common cation in acid medium, with results pointing to approximately 90% population of the imino species for 1-mOH(4)C and 1,5-m(2)OH(4)C, further supported by NMR spectroscopy. Both exhibited two sequential dissociations in alkaline medium, the first due to N(4)-OH, followed by the N(3)-H. (1)H and (13)C NMR spectroscopy showed 1-mOH(4)C in the conformation syn. With 1,3,5-m(3)OH(4)C, an ;overcrowded' planar molecule with steric constraints to both the syn and anti conformations, a syn-anti equilibrium is observed, with a preference of approximately 75% for the anti rotamer, independently of the polarity of the medium. Exchange between the rotamers is slow on the NMR time-scale, with a minimal barrier to exchange exceeding 100 kJ/mol. In low-polar media, the analogues associate as dimers via O(4)-Hcdots, three dots, centeredO(2) or O(4)-Hcdots, three dots, centeredN(4) hydrogen bonds, with association constants at ambient temperature of 4.6 (1,3-m(2)OH(4)C), 12.8 (anti 1,3,5-m(3)OH(4)C), 36 (1,5-m(2)OH(4)C), 109 (syn 1,3,5-m(3)OH(4)C) M(-1). Implications of the overall findings to the promutagenic activities of OH(4)C and OMe(4)C are examined.

Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues.

Intramolecular stacking of a series of new synthesized dinucleotide mRNA cap analogues has been investigated in aqueous buffers by means of fluorescence and 1H-NMR at various pH and temperatures, and compared with that for 7-methylguanosine(5')ppp(5')guanosine (m7GpppG), as well as its hypermethylated derivative m(3)2,2,7GpppG. Thermodynamic parameters for intramolecular self-association stabilized by stacking were established by temperature-dependent fluorescence quenching, taking into account collisional deactivation of the excited states. Relative orientations of the stacked bases in the cap analogues were determined with the aid of a program GEOSHIFT (Stolarski et al., Biochim. Biophys. Acta (1996) 1293, 97), based on ring-current anisotropy. 1D-soft-TOCSY experiments were applied to extract the exact values of vicinal coupling constants, and hence to resolve solution conformation of the cap molecules. Stacking interaction has been discussed in detail in terms of the cap structural features, e.g., types of bases and length of the 5',5'-phosphate bridges, and regarding the interactions stabilizing intramolecular stacking.

Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: ligand-dependent quenching of enzyme intrinsic fluorescence.

Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E. coli purine nucleoside phosphorylase (PNP) with phosphate (Pi; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluorescence by Pi was bimodal, best described by two modified Stem-Volmer equations fitted independently for "low" (below 0.5 mM Pi) and "high" (above 0.5 mM Pi) ligand concentrations. At Pi > 0.5 mM, binding is characterized by a fortyfold higher dissociation constant (Kd2 = 1.12 +/- 0.10 mM), i.e. by a lower affinity for phosphate, with a sevenfold lower quenching constant and 1.6-fold higher accessibility. By contrast, the binding of SA, and the resultant fluorescence quenching, was unimodal, with Kd = 1.36 +/- 0.07 mM, comparable to the Kd for Pi at "high" Pi, with a total binding capacity of one sulfate or phosphate group per enzyme subunit. SA proved to be a competitive inhibitor of phosphorolysis with Ki = 1.2 +/- 0.2 mM vs. Pi, hence similar to its Kd. SA at a concentration of 5 mM did not affect the Pi affinity at Pi < 0.5 mM, but led to a reduced affinity and twofold higher Pi binding capacities at Pi > 0.5 mM. The resultant fluorescence quenching by Pi decreased at 5 mM SA, with lower Stern-Volmer constant (KSV) and fractional accessibility (fa) values. Increasing concentrations of Pi reduced the enzyme affinity for SA, characterized by a higher Kd. The Hill model showed negative cooperative binding of Pi in the absence and presence of 5 mM SA with Hill coefficients h = 0.60 +/- 0.01 and h = 0.83 +/- 0.07, respectively. SA exhibited non-cooperative binding in the absence of Pi (h = 1.08 +/- 0.01) and negative cooperative binding in the presence of Pi (h < 1). PNP fluorescence decays were best fitted to a sum of two exponentials, with an average lifetime of 2.40 +/- 0.14 ns unchanged on interaction with quenching ligands, and pointing to static quenching. The overall results are relevant to the properties of PNP from various sources, in particular to the design of potent bisubstrate analogue inhibitors.

Fluorescence of reduced nicotinamides using one- and two-photon excitation.

We examined the steady-state and time-resolved emission of NADH and NAMH resulting from one-photon and two-photon excitation. Similar emission spectra were observed for both modes of excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690 to 740 nm, which is 46% higher than the value of 0.37 observed for one-photon excitation. This observation of a higher anisotropy with two-photon excitation was consistent with INDO/SDCI calculations of the one- and two-photon transitions. Minor differences in the multi-exponential decays of NADH were observed for one- and two-photon excitation, but presently available resolution does not allow us to conclude the decays are distinct. NADH-LADH-IBA complex formation led to an order of magnitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluorescence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluorescence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interactions with adenine part of NADH. Such interactions are responsible for the depolarization of NADH fluorescence observed for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excitation which suggests that fluorescence from NADH will be moderately difficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation.

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r 0) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-L-tryptophanamide.

Fluorescence anisotropy of tyrosine using one-and two-photon excitation.

We examined the emission spectra and steady-state anisotropy of tyrosyl fluorescence with two-photon excitation from 565 to 578 nm. The emission spectra of phenol and N-acetyl-L-tyrosinamide (NATyrA) were all the same for one-photon excitation (OPE) and two-photon excitation (TPE), and the tyrosine emission from ribonuclease A showed 10-nm shift to longer wavelengths with TPE. Surprisingly, the anisotropy of tyrosine, NATyrA and Leu5-enkephalin in frozen solution were near zero for TPE as compared to near 0.3 for OPE. Low values of the anisotropy near 0.05 were also found for phenol and ribonuclease A. A low anisotropy appears to be a basic characteristic of tyrosine or tyrosyl residues with two-photon excitation.

Nucleoside triphosphate donors for nucleoside kinases: donor properties of UTP with human deoxycytidine kinase.

The reported higher efficiency of UTP, relative to ATP, as phosphate donor for deoxycytidine kinase (dCK), has been extended and found to apply to both dCyd and dAdo as acceptors. UTP as phosphate donor was shown to follow strictly Michaelis kinetics, with Km = 1 microM, in striking contrast to ATP, which exhibits marked negative cooperativity (Hill coef. = 0.7) with a several-fold higher Kmapp = 15 microM. Phosphate transfer was followed directly with use of mixtures of [gamma-32P]ATP and cold UTP as donors, or with 3H-labeled acceptors and cold donors. With equimolar concentrations of ATP and UTP (50 microM or 1 mM each), and dCyd or dAdo as acceptor, only minimal phosphate transfer occurred from ATP (3-10%). With a 6:1 ratio of ATP:UTP, hence exceeding the intracellular ratio, phosphate transfer from ATP increased, but still did not exceed 25-40% with either dCyd or dAdo as acceptor. Moreover, relative ATP transfer is dependent on the dCyd concentration. We conclude that the major intracellular phosphate donor for dCK is not ATP, but UTP. Preliminary data for human thymidine kinases (TK1 and TK2) exhibit quite different behaviour. The foregoing, together with literature data, are highly relevant to in vitro studies on the properties of the nucleoside kinases, and to the design of chemotherapeutically active nucleoside analogues.

Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation.

We examined the emission spectra of tyrosine- and tryptophan-containing proteins using one-photon (270-310 nm) and two-photon (565-610 nm) excitation. Emission spectra for two-photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one-photon excitation below 290 nm. We examined the one-photon and two-photon excitation spectra of tyrosine-tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short-wavelength shift of the tyrosine two-photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett. 208, 276-282.

Binding of substrates to human deoxycytidine kinase studied with ligand-dependent quenching of enzyme intrinsic fluorescence.

Deoxycytidine kinase is a key enzyme in the salvage pathway, and its activity is required for 5'-phosphorylation of several important antiviral and cytostatic nucleoside analogues. It has recently been purified completely from human sources. Steady-state and time-resolved fluorescence of human deoxycytidine kinase was used to study its interaction with the substrates dCyd, dAdo, dUrd, dTTP, and the feedback inhibitor dCTP. Enzyme fluorescence quenching by dCTP, dCyd, dTTP, and dAdo was bimodal, and the best fits of the quenching patterns were obtained using two modified Stern-Volmer equations with two sets of quenching constants (Ksv) and accessibility values (fa) fitted independently for "low" and "high" concentration ranges of ligands. The transition between these occurred at about 20 microM dCTP, 50 microM dCyd, 30 microM dTTP, and 180 microM dAdo. Enzyme fluorescence showed unimodal quenching by dAdo and 30% reduced accessibility of the binding site in the presence of dCyd. dUrd quenching was also unimodal with Ksv = 0.0047 +/- 0.0007 microM-1 and fa = 0.75 +/- 0.05, hence in the same range as for the "high" concentration range of dAdo in the absence of dCyd, where they are 0.0025 +/- 0.0003 microM-1 and 0.73 +/- 0.03, respectively. Fluorescence quenching was used to directly determine enzyme-ligand binding and revealed bimodal binding of dCTP, dCyd, dTTP, and dAdo and unimodal binding of dUrd, and of dAdo in the presence of 0.1 microM dCyd. Transition between these two modes of binding occurred at the concentrations described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of human deoxycytidine kinase toward antiviral 2',3'-dideoxynucleoside analogs.

Deoxycytidine (dCyd) kinase has been purified to homogeneity from human leukemic spleen, and the capacity of the enzyme to phosphorylate 2',3'-dideoxynucleoside (ddN) analogs that are clinically effective inhibitors of human immunodeficiency virus (HIV) replication was evaluated. Cytosine-containing ddN analogs, such as 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-dehydrocytidine, and cytallene, were efficiently phosphorylated by dCyd kinase, while no phosphorylation of purine-containing ddN analogs was detected. dCyd kinase was completely inactive toward 2',3'-dideoxyadenosine (ddAdo), 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and adenallene, although it was capable of phosphorylating both 2'-deoxyadenosine (dAdo) and 2'-deoxyguanosine (dGuo). The abilities of wild type and mutant human T lymphoblastoid CEM cells to accumulate ddAdo in situ and in vitro were also ascertained. Comparison of the abilities of intact wild type CEM cells and derivatives deficient in nucleoside transport, dCyd kinase, and/or adenosine (Ado) kinase to accumulate [3H]ddAdo-derived radioactivity revealed no significant differences among the wild type and mutant strains. However, ddAdo phosphorylating activity was decreased in extracts from Ado kinase-deficient cells but not in lysates prepared from cells genetically deficient in dCyd kinase activity. In comparative growth rate experiments, wild type, nucleoside transport-deficient, and dCyd kinase-deficient CEM cells were equally sensitive to ddAdo toxicity, while, interestingly, a deficiency in Ado kinase correlated with a 5-fold decreased growth sensitivity to the purine ddN. Insertion of an adenine phosphoribosyltransferase deficiency into the CEM cell lines did not influence ddAdo toxicity or incorporation rate. These results imply that Ado kinase may be an important factor in ddAdo phosphorylation by CEM cells. Furthermore, these studies demonstrate that cytosine- and purine-containing ddNs are transported and activated by independent pathways and, therefore, have important implications for anti-HIV therapy in that pyrimidine and purine ddNs might be used in combination for the treatment of acquired immunodeficiency syndrome.

Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs.

Deoxynucleoside kinases are required for the 5'-phosphorylation of deoxynucleoside analogs used in chemotherapy. Cytoplasmic thymidine kinase (TK1), deoxycytidine kinase (dCK) and mitochondrial thymidine kinase (TK2) were completely purified from human leukemic spleen and their capacities to phosphorylate 43 nucleoside analogs were compared. TK1 showed the most restricted substrate specificity but tolerated 3'-modifications of the sugar ring and some 5-substitutions of the pyrimidine ring. TK2 showed a much broader specificity and phosphorylated pyrimidine bases with bulky 5-substitutions, including cytosine analogs, while sugar analogs with substituents other than OH in the 2' and 3' positions were very poor substrates. dCK showed a very broad specificity phosphorylating several cytosine analogs with 2' and 3' modifications as well as acyclic sugar analogs. Purine deoxyribonucleosides were also efficiently phosphorylated by dCK but in this case sugar modifications led to drastically decreased activity.