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Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani
Molecular and Biochemical Parasitology, 28 (1988) 181-188 Elsevier
181
MBP 00947
Reaction kinetics and inhibition of adenosine kinase from Leishmania
donovani Dipa Bhaumik and Alok K. Datta Leishmania Group, Indian Institute of Chemical Biology, Calcutta, India (Received 28 August 1987; accepted 4 December 1987)
The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2--ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [~/-32p]ATP resulted in binding of adenosine to free enzyme. Tubercidin (7-deazaadenosine) and 6-methylmercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vm,x/K~)' and 'turnover numbers (Kc,t)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme. Key words: Adenosine kinase; Enzyme kinetics; Nucleoside analog phosphorylation; Leishmania donovani
Introduction
Most parasitic protozoa, including Leishmania donovani, scavenge preformed host purines utilizing their salvage pathway enzymes [1-7]. Mutant cells of L. donovani lacking adenosine kinase, a purine salvage enzyme, metabolize adenosine at rates 25% of that observed in wild type cells [8]. This observation, coupled with the finding that adenosine kinase activity is stimulated several fold during transformation from promastigotes to amastigotes [9] led to extensive investigations on the role of the enzyme in aden-
Correspondence address: A.K. Datta, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700032, India. Abbreviations: DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrile)]tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride.
osine metabolism and its transport during parasite multiplication [10,11]. As a part of this effort we undertook a detailed study on the kinetic properties and inhibitor specificities of the enzyme. Our previous studies [12] demonstrated unique characteristics of adenosine kinase from L. donovani. Tubercidin and 6-methylmercaptopurine riboside, the two cytotoxic isomers of adenosine, are phosphorylated by mammalian adenosine kinase [13,14]. Resistance to tubercidin and 6-methylmercaptopurine riboside is associated with a genetic deficiency in adenosine kinase activity [15-18]. These two analogs also inhibit L. donovani and other parasite multiplications (D.B., unpublished data) [19]. Reports from various laboratories have implicated adenosine kinase as the enzyme responsible for the initial step of monophosphorylation of these adenosine analogs in parasitic protozoa [8,20]. Most of these conclusions are circumstantial and extrapolations from
0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
182
mammalian systems. These extrapolations may not always be correct due to the fact that in Schistosoma mansoni and Giardia lamblia adenosine kinase is hardly detectable. Nevertheless, these parasites are sensitive to tubercidin and other adenosine analogs [21,22]. In this communication using a homogeneous preparation of L. donovani adenosine kinase we provide direct evidence to show that tubercidin and 6-methylmercaptopurine riboside are substrates for the enzyme and both bind at the adenosine binding site of the enzyme. Materials and Methods
Reagents. [2,8-3H]Adenosine (32 Ci mmol 1) was obtained from New England Nuclear (Boston, MA). [',/-32p]ATP (3000 Ci mmol 1) was the product of Amersham International (Buckinghamshire, U.K.). Tubercidin, 6-methylmercaptopurine riboside and the other nucleosides and nucleotides were the products of Sigma Chemical Co. (St. Louis, MO). Pyruvate kinase and lactate dehydrogenase were from Boehringer (Mannhelm). All other chemicals used were of reagent grade. The purity of the nucleotides was checked by paper chromatography. Enzyme preparation. Adenosine kinase used in these studies was purified approximately 3300 fold from L. donovani (MHOM/IN/1978/UR6), a strain isolated from an Indian kala-azar patient [12,23]. Enzyme assays and initial velocity determinations. Radiochemical and spectrophotometric assays were carried out following the procedure as described elsewhere [12]. For initial velocity studies and competition experiments assays were carried out by radiochemical method while determination of kinetic constants of individual nucleoside/analogs was done by spectrophotometric assay. The linearity of all plots was checked graphically for all substrate concentrations. For determination of initial velocities of the reactions, kinetic data were fitted into MichaelisMenten equation, i.e., rectangular hyperbola and double reciprocal plots were done according to Cleland [24]. Secondary plots were used to de-
termine K m and Ki values.
Equilibrium dialysis. In two separate experiments equilibrium dialysis of the purifed enzyme (0.2 ml, 5.1 t~g ml -t) were carried out for 68 h against 20 mM potassium phosphate pH 7.5, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM [ethylenebis(oxyethylenenitrile)]tetraacetic acid (EGTA), 1 mM each of benzamidine-HCl, iodoacetic acid, phenylmethylsulfonyl fluoride (PMSF) and 5% glycerol containing [3H]adenosine (32 Ci mmol 1) and ['y-32p]ATP (3000 Ci mmol 1). After dialysis, radioactivity inside and outside the bag was measured using Bray's scintillation fluid. Results
Stoichiornetry of reaction. The studies on the stoichiometry of adenosine kinase reaction revealed that 1 mol of AMP is formed during the hydrolysis of 1 tool of ATP to ADP (results not presented). Initial velocity pattern. When the concentrations of adenosine or M g > - A T P were varied in turn at a series of fixed concentrations of the second substrate, double reciprocal plots of initial velocity displayed a family of intersecting lines converging at a point left of the vertical axis and below the horizontal axis (Fig. 1). This is typical of bisubstrate reactions undergoing sequential mechanism involving ternary enzyme-substrate complex [24]. It cannot be said, however, from these results whether the binding of the substrates to the enzyme is ordered or random. Secondary plots of the slope and intercept produced a K m for adenosine (Fig. 1A, inset) and MgZ+-ATP (Fig. IB, inset) of 26.0 and 71.4 I~M respectively. Product inhibition studies. L. donovani enzyme is inhibited by the reaction products AMP and ADP. In order to identify first and second substrates and to determine their sequence of addition, product inhibition studies were carried out by varying one of the substrates while holding the second substrate at saturating levels. Mg > concentrations in the reaction were maintained at a fixed excess over the nucleotides present at the
183 competitive inhibition observed with variable adenosine concentration changed to uncompetitive nature when the concentration of A T P was raised to a value of about 100 times the K,n of A T P (results not presented). These results are compatible with the general trend of ordered BiBi reaction mechanism in which adenosine appears to be the first substrate to bind and AMP the last product to be released from the enzyme molecule [24,25].
initiation of the reaction. Results presented in Fig. 2 show that A M P is a competitive inhibitor with respect to adenosine (Fig. 2A) having a K i (slope) of about 320 p~M. Secondary plot of the slope is linear (Fig. 2A, inset). In contrast, A M P behaves as a noncompetitive inhibitor in presence of varying concentrations of A T P - M g 2+ with Ki (slope) and K i (intercept) of about 146 and 560 p~M respectively. When inhibition by A D P at varying concentrations of adenosine and ATP-Mg =+ was evaluated in two parallel experiments keeping the concentration of second substrate at just saturating levels, the double reciprocal plots in both cases revealed a noncompetitive nature of inhibition (Fig. 3). In both the cases slope and intercept replots are linear with K i (slope) and K i (intercept) values as 1040 p~M and 906 p~M respectively for the experiments involving saturating ATP-Mg 2+ and variable adenosine (Fig. 3A), whereas Ki (slope) and Ki (intercept) were determined as 160 and 240 ~M respectively for experiments involving saturating adenosine and varying ATP-Mg 2+ (Fig. 3B). It should be mentioned here that the non-
8.0 IA | --" 7.0
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50
allel equilibrium dialysis experiments against [3H]adenosine and [~/-32P]ATP resulted in binding of free adenosine to the enzyme molecule. There was no binding of A T P onto the enzyme. Assuming the molecular weight of enzyme as 38000 [12], approximate molar stoichiometry of binding of enzyme to adenosine was determined as 1:0.16.
Inhibition by adenosine analogs. Phosphorylation of [3H]adenosine by L. donovani adenosine kinase is inhibited by tubercidin and 6-methylmer-
~- EAdo]pM I 0.2
To .oL O.O ,, '~
Direct demonstration of adenosine binding. Par-
~
]'~
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05
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2.0
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~> 1.0 i
0.1 I/[ Mg-ATP]p~l
1
0.2
I
I
0.5 l/EAdenosine] p~1
I
1.0
Fig. 1. (A) Double reciprocal plots of initial velocity with variable ATP concentrations at several fixed concentrations of adenosine. Inset shows secondary plots of the slope (o--e) and the intercept (o--o) against inverse of adenosine concentration. (B) Double reciprocal plots of initial velocitywith variable adenosine concentration at several fixed concentrationsof ATP. Inset shows secondary plots of the slope (e--e) and the intercept (o--o) against inverse of [ATP-Mg2-] concentration.
184
I2] A ,
EAMP]pM..o
,_-;
'~'°~°1 ,
,
.0
oo/
/
i
B
~ [AMP]pM
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/'
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~o/
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-6 0.4 E C
to 0 0.2 x ::> --,.,
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0.5
0.4
0.01
0.02
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I
0.05
1
0.04
I/[Mg-ATP] ~ I
Fig. 2. Double reciprocal plots of AMP inhibition with variable adenosine (A) and ATP (B) concentrations. Insets display slope ( e - - o ) and intercept (o--o) replots versus AMP concentration.
.
A
EMg- ADP-IPMjb
B
EMg-ADP] #M
1.2
%,
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1.0
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52,0 Tu, E
~0~~
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E cl
-I.0 c
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0.Sx
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0.4
0.02
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0.04
>
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I / [ M g - A T P ] 1.1[~I
Fig. 3. Double reciprocal plots of ADP inhibition with variable adenosine (A) and ATP (B) concentrations. Slope ( o - - e ) and intercept (o--o) replots are displayed against ADP concentration.
185 TABLE I Kinetic constants of adenosine and its analogs Kinetic constants ~
Adenosine/analogs
Adenosine Tubercidin 6-Methylmercaptopurine riboside
v,~x
Km
Vmax/Km
/':cat
K,,/gm
(Ixmol min 1 rag-<)
(IxM)
(1 min -l mg 1)
(s-l)
(s -1 txM l)
38.5 37 22.7
16 4.8 2.6
2.4 7.7 8.7
24 23 14
1.5 4.8 5.4
a Vm~xand Kr~ax determinations were done by spectrophotometric assay as described in Materials and Methods.
captopurine riboside [12]. Detailed studies indicate that both the analogs, besides being substrates themselves, behave as competitive inhibitors of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. The K i (slope) for tubercidin and 6-methylmercaptopurine riboside were determined as 70 IxM. Table I depicts composite kinetic constants of these compounds computed from spectrophotometric assay. Discussion
The results of the kinetic studies and equilibrium dialysis experiment are consistent with an ordered Bi-Bi addition of substrate and release of products with adenosine being the first substrate to bind and AMP the last product to be released. Schematically it may be shown as in Fig. 4. Our studies are in agreement with the kinetic properties of the enzyme from human placenta [26] but differ from the results obtained with enzymes from Ehrlich ascites tumor [27] and murine leukemia L1210 cell [28] lines with regard to order and sequence of addition of substrates. The kinetic properties and values for the kinetic constants of L. donovani adenosine kinase may be related to in vivo regulation of the enzyme. The phenomenon of adenosine kinase in-
Ad
ATP
1 E~--~E
ADP
T
Ad _
Ad ~ c-AMP EATP .~-----F_ADP ~
AMP
".c-AMP F
[
"E
Fig. 4. Reaction mechanism for L. donovani adenosine kinase.
hibition by AMP and ADP is interesting for several reasons. K~ values for AMP and ADP are in the range of their intracellular concentrations [29]. These inhibitions are consistent with the concept of regulation by ATP 'charge' as proposed by Atkinson and Fall [30]. The hypothesis proposed by them provides for ATP conservation within the cell so that the rate of reactions utilizing ATP is decreased when the ratio of [ATP]/[AMP] +[ADP] becomes low. Concentration of ATP has been shown to be about 50 times greater in mouse L-cells than in L. donovani [31]. Thus, the possibility of a correlation between the level of ATP and phosphorylating ability of adenosine kinase within L. donovani cells is not unlikely. Regarding the inhibitor and substrate specificity of L. donovani adenosine kinase it is interesting to note that both tubercidin and 6-methylmercaptopurine riboside are inhibitors of adenosine phosphorylation in vitro and parasite multiplication as well [8,19]. The studies indicate that at 50 txM substrate concentrations of each, reaction rates are in the order tubercidin> adenosine> 6-methylmercaptopurine riboside [12] whereas determination of Vma×/Km and Kcat/K m values fall in the sequence 6-methylmercaptopurine riboside>tubercidin>adenosine. This observation, together with other reports on the intracellular concentration of adenosine in eukaryotic cells (1 txM) [32,33], indicate that the efficacy of the compounds in vivo would be determined by the intracellular pool of adenosine. The results also suggest that in the absence of phosphotransferase activity in L. donovani [9] phosphorylation of nucleosides and their analogs is carried out mostly, if not entirely, by adenosine kinase. Furthermore, the observation that adenosine can be directly metabolized through three
186
different pathways in L. donovani, namely, phosphorylation, deamination and phosphorolysis [9], indicates that the K m value for adenosine with respect to adenosine kinase, adenosine deaminase and adenosine phosphorylase should determine which pathway will be favored in the presence of the substrate analogs. Thus the existence of multiple purine salvage pathways in L. donovani [3] indicates that perhaps in no case would inhibition of adenosine kinase alone be lethal unless other enzymes of salvage pathway are selectively targeted. Nevertheless, the observation reported
here could be useful while studying the role of this enzyme during parasite multiplication. Acknowledgements The authors wish to thank Mr. H.N. Dutta and his associates for art work. Thanks are also due to Drs. A.N. Bhaduri and Samit Adhya of this Institute for careful reading of the manuscript. D.B. is supported by a Junior Research Fellowship from the indian Council of Medical Research, New Delhi, India.
References 1 Gutteridge, W.E. and Coombs, G.H. (1977) Biochemistry of Parasitic Protozoa, University Park Press, Baltimore. 2 Walsh, C.J. and Sherman, I.W. (1968) Purine and pyrimidine synthesis by the avian malarial parasite Plasmodium lophurae. J. Protozool. 15,763-770. 3 Marr, J.J., Berens, R.L. and Nelson, D.J. (1978) Purine metabolism in Leishmania donovani and Leishmania braziliensis. Biochim. Biophys. Acta 544, 360-371. 4 Ceron, C.R., Caldas, R.A., Felix, C.F., Mundim, M.H. and Roitman, I. (1979) Purine metabolism in trypanosomatids. J. Protozool. 26, 479-483. 5 K6nigk, E. and Putfarkan, B. (1980) Stage-specific differences of a perhaps signal-transferring system in Leishmania donovani. Tropenmed. Parasitol. 31,421-424. 6 K6nigk, E. (1978) Purine nucleotide metabolism in promastigotes of Leishmania tropica: inhibitory effect of allopurinol and analogues of purine nucleosides. Tropenmed. Parasitol. 29, 435-438. 7 Lafon, S.W., Nelson, D.S., Berens, R.L. and Mart, J.J. (1982) Purine and pyrimidine salvage pathways in Leishmania donovani. Biochem. Pharmacol. 31,231-238. 8 Iovannisci, D.M. and Ullman, B. (1984) Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity. Mol. Biochem. Parasitol. 12, 139 151. 9 Looker, D.L., Berens, R.L. and Mart, J.J. (1983) Purine metabolism in Leishmania donovani amastigotes and promastigotes. Mol. Biochem. Parasitol. 9, 15 28. 10 Iovannisci, D.M., Kaur, K., Young, L. and Ullman, B. (1984) Genetic analysis of nucleoside transport in Leishmania donovani. Mol. Cell. Biol. 4, 1013-1019. 11 Aronow, B., Kaur, K., McCartan, K. and Ulhnan, B. (1987) Two high affinity nucleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22, 29-37. 12 Datta, A.K., Bhaumik, D. and Chatterjee, R. (1987) Isolation and characterization of adenosine kinase from Leishmania donovani. J. Biol. Chem. 262, 5515-5521. 13 Lindberg, B., Klenow, H. and Hansen, K. (1967) Some properties of partially purified mammalian adenosine kinase. J. Biol. Chem. 242, 350~356. 14 Divekar, A.Y. and Hakala, M.T. (1971) Adenosine kinase of sarcoma 180 cells (N~'-substituted adenosines as
substrates and inhibitors). Mol. Pharmacol. 7,663-673. 15 Gudas, L.J., Cohen, A., Ullman, B. and Martin, D.W. Jr. (1978) Analysis of adenosine mediated pyrimidine starvation using cultured wild type and mutant mouse T-lymphoma cells. Somatic Cell Genet. 4,201 219. 16 Divekar, A.Y., Kenny, L.N. and Hakala, M.T. (1971) Changes in Sarcoma-180 (S-180) cells associated with resistance to adenosine (AR) analogs. Fed. Proc. 30, 1253. 17 Bennett, L.L., Jr., Brockman, R.W., Schnebli, H.P., Chumley, S., Dixon, G.J., Schabel, F.H., Skipper, H.E., Montgomery, J.A. and Thomas, J. (1965) Activity and mechanism of action of 6-methylthiopurine riboside in cancer cells resistant to 6-mercaptopurine. Nature 205, 1276-1279. 18 Ho, D,H.W.. Luce, J.K. and Frei, E., [lI (1968) Distribution of purine ribonucleoside kinase and selective toxicity of 6-methylpurine ribonucleoside. Biochem. Pharmacol. 17, 1025-1035. 19 Acs, G., Reich, E. and Mori, M. (1964) Biological and biochemical properties of the analogue antibiotic tubercidin. Proc. Natl. Acad. Sci. USA 52,493-501. 20 Schwartzman, J.D. and Pferferkorn, E.R. (1982) Toxoplasma gondii purine synthesis and salvage in mutant host cells and parasites. Exp. Parasitol. 53, 77-86. 21 Dovcy, H.F., Mckerrow, J.H, and Wang, C.C. (1985) Action of tubercidin and other adenosine analogs in Schistosorna mansoni schistosomules. Mol. Biochem. Parasitol. 16, 185 198. 22 Berens, R.L. and Marr, J.J. (1986) Adenosine analog metabolism in Giardia lamblia. Biochem. Pharmacol. 35, 4191-4197. 23 Ghosh, A.K., Bhattacharyya, F.K. and Ghosh, D.K. (1985) Leishmania donovani amastigote inhibition and mode of action of berberine. Exp. Parasitol. 60,404-413. 24 Cleland, W.W. (1970) Steady state kinetics. In: The Enzymes (Boyer, P.D., ed.) 3rd edn. w)l. 2, pp. 1-65, Academic Press, New York. 25 Cleland, W.W. (1963) The kinetics of enzyme catalysed reactions with two or more substrates or products. Biochim. Biophys. Acta 67, 104-137. 26 Palella, T.D., Andres, C.M. and Fox, I.H. (1980) Human
187
27
28
29 30
placental adenosine kinase (kinetic mechanism and inhibition). J. Biol. Chem. 255, 5264-5269. Henderson, J.F., Mikoshiba, A., Chu, S.Y. and Caldwall, I.C. (1972) Kinetic studies of adenosine kinase from Ehrlich ascites tumor cells. J. Biol. Chem. 247, 1972-1975. Chang, C-H, Cha, S., Brockman, R.W. and Bennett, L.L. Jr. (1983) Kinetic studies of adenosine kinase from L1210 cells: A model enzyme with a two-site ping-pong mechanism. Biochemistry 22, 600-611. Bartlett, G.R. (1968) Phosphorous compounds in the human erythrocyte. Biochim. Biophys. Acta 156, 221-230. Atkinson, D.E. and Fall, I. (1967) Adenosine triphosphate conservation in biosynthetic regulation. J. Biol.
Chem. 242, 3241-3242. 31 Lafon, S.W., Nelson, D.J., Berens, R.L. and Mart, J.J. (1985) Inosine analogs, their metabolism in mouse L cells and in Leishmania donovani. J. Biol. Chem. 260, 9660-9665. 32 Fox, A.C., Reed, G.E., Glassman, E., Kaltman, A.J. and Silk, B.B. (1974) Release of adenosine from human hearts during angina induced by rapid atrial pacing. J. Clin. Invest. 53, 1447-1457. 33 Mills, G.C., Schmalsteig, F.C., Trimmer, K.B., Goldman, A.S. and Goldblum, R.M. (1976) Purine metabolism in an adenosine deaminase deficiency. Proc. Natl. Acad. Sci. USA 73, 2867-2871.
181
MBP 00947
Reaction kinetics and inhibition of adenosine kinase from Leishmania
donovani Dipa Bhaumik and Alok K. Datta Leishmania Group, Indian Institute of Chemical Biology, Calcutta, India (Received 28 August 1987; accepted 4 December 1987)
The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2--ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [~/-32p]ATP resulted in binding of adenosine to free enzyme. Tubercidin (7-deazaadenosine) and 6-methylmercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vm,x/K~)' and 'turnover numbers (Kc,t)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme. Key words: Adenosine kinase; Enzyme kinetics; Nucleoside analog phosphorylation; Leishmania donovani
Introduction
Most parasitic protozoa, including Leishmania donovani, scavenge preformed host purines utilizing their salvage pathway enzymes [1-7]. Mutant cells of L. donovani lacking adenosine kinase, a purine salvage enzyme, metabolize adenosine at rates 25% of that observed in wild type cells [8]. This observation, coupled with the finding that adenosine kinase activity is stimulated several fold during transformation from promastigotes to amastigotes [9] led to extensive investigations on the role of the enzyme in aden-
Correspondence address: A.K. Datta, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700032, India. Abbreviations: DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrile)]tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride.
osine metabolism and its transport during parasite multiplication [10,11]. As a part of this effort we undertook a detailed study on the kinetic properties and inhibitor specificities of the enzyme. Our previous studies [12] demonstrated unique characteristics of adenosine kinase from L. donovani. Tubercidin and 6-methylmercaptopurine riboside, the two cytotoxic isomers of adenosine, are phosphorylated by mammalian adenosine kinase [13,14]. Resistance to tubercidin and 6-methylmercaptopurine riboside is associated with a genetic deficiency in adenosine kinase activity [15-18]. These two analogs also inhibit L. donovani and other parasite multiplications (D.B., unpublished data) [19]. Reports from various laboratories have implicated adenosine kinase as the enzyme responsible for the initial step of monophosphorylation of these adenosine analogs in parasitic protozoa [8,20]. Most of these conclusions are circumstantial and extrapolations from
0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
182
mammalian systems. These extrapolations may not always be correct due to the fact that in Schistosoma mansoni and Giardia lamblia adenosine kinase is hardly detectable. Nevertheless, these parasites are sensitive to tubercidin and other adenosine analogs [21,22]. In this communication using a homogeneous preparation of L. donovani adenosine kinase we provide direct evidence to show that tubercidin and 6-methylmercaptopurine riboside are substrates for the enzyme and both bind at the adenosine binding site of the enzyme. Materials and Methods
Reagents. [2,8-3H]Adenosine (32 Ci mmol 1) was obtained from New England Nuclear (Boston, MA). [',/-32p]ATP (3000 Ci mmol 1) was the product of Amersham International (Buckinghamshire, U.K.). Tubercidin, 6-methylmercaptopurine riboside and the other nucleosides and nucleotides were the products of Sigma Chemical Co. (St. Louis, MO). Pyruvate kinase and lactate dehydrogenase were from Boehringer (Mannhelm). All other chemicals used were of reagent grade. The purity of the nucleotides was checked by paper chromatography. Enzyme preparation. Adenosine kinase used in these studies was purified approximately 3300 fold from L. donovani (MHOM/IN/1978/UR6), a strain isolated from an Indian kala-azar patient [12,23]. Enzyme assays and initial velocity determinations. Radiochemical and spectrophotometric assays were carried out following the procedure as described elsewhere [12]. For initial velocity studies and competition experiments assays were carried out by radiochemical method while determination of kinetic constants of individual nucleoside/analogs was done by spectrophotometric assay. The linearity of all plots was checked graphically for all substrate concentrations. For determination of initial velocities of the reactions, kinetic data were fitted into MichaelisMenten equation, i.e., rectangular hyperbola and double reciprocal plots were done according to Cleland [24]. Secondary plots were used to de-
termine K m and Ki values.
Equilibrium dialysis. In two separate experiments equilibrium dialysis of the purifed enzyme (0.2 ml, 5.1 t~g ml -t) were carried out for 68 h against 20 mM potassium phosphate pH 7.5, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM [ethylenebis(oxyethylenenitrile)]tetraacetic acid (EGTA), 1 mM each of benzamidine-HCl, iodoacetic acid, phenylmethylsulfonyl fluoride (PMSF) and 5% glycerol containing [3H]adenosine (32 Ci mmol 1) and ['y-32p]ATP (3000 Ci mmol 1). After dialysis, radioactivity inside and outside the bag was measured using Bray's scintillation fluid. Results
Stoichiornetry of reaction. The studies on the stoichiometry of adenosine kinase reaction revealed that 1 mol of AMP is formed during the hydrolysis of 1 tool of ATP to ADP (results not presented). Initial velocity pattern. When the concentrations of adenosine or M g > - A T P were varied in turn at a series of fixed concentrations of the second substrate, double reciprocal plots of initial velocity displayed a family of intersecting lines converging at a point left of the vertical axis and below the horizontal axis (Fig. 1). This is typical of bisubstrate reactions undergoing sequential mechanism involving ternary enzyme-substrate complex [24]. It cannot be said, however, from these results whether the binding of the substrates to the enzyme is ordered or random. Secondary plots of the slope and intercept produced a K m for adenosine (Fig. 1A, inset) and MgZ+-ATP (Fig. IB, inset) of 26.0 and 71.4 I~M respectively. Product inhibition studies. L. donovani enzyme is inhibited by the reaction products AMP and ADP. In order to identify first and second substrates and to determine their sequence of addition, product inhibition studies were carried out by varying one of the substrates while holding the second substrate at saturating levels. Mg > concentrations in the reaction were maintained at a fixed excess over the nucleotides present at the
183 competitive inhibition observed with variable adenosine concentration changed to uncompetitive nature when the concentration of A T P was raised to a value of about 100 times the K,n of A T P (results not presented). These results are compatible with the general trend of ordered BiBi reaction mechanism in which adenosine appears to be the first substrate to bind and AMP the last product to be released from the enzyme molecule [24,25].
initiation of the reaction. Results presented in Fig. 2 show that A M P is a competitive inhibitor with respect to adenosine (Fig. 2A) having a K i (slope) of about 320 p~M. Secondary plot of the slope is linear (Fig. 2A, inset). In contrast, A M P behaves as a noncompetitive inhibitor in presence of varying concentrations of A T P - M g 2+ with Ki (slope) and K i (intercept) of about 146 and 560 p~M respectively. When inhibition by A D P at varying concentrations of adenosine and ATP-Mg =+ was evaluated in two parallel experiments keeping the concentration of second substrate at just saturating levels, the double reciprocal plots in both cases revealed a noncompetitive nature of inhibition (Fig. 3). In both the cases slope and intercept replots are linear with K i (slope) and K i (intercept) values as 1040 p~M and 906 p~M respectively for the experiments involving saturating ATP-Mg 2+ and variable adenosine (Fig. 3A), whereas Ki (slope) and Ki (intercept) were determined as 160 and 240 ~M respectively for experiments involving saturating adenosine and varying ATP-Mg 2+ (Fig. 3B). It should be mentioned here that the non-
8.0 IA | --" 7.0
/
i.o oJ
50
allel equilibrium dialysis experiments against [3H]adenosine and [~/-32P]ATP resulted in binding of free adenosine to the enzyme molecule. There was no binding of A T P onto the enzyme. Assuming the molecular weight of enzyme as 38000 [12], approximate molar stoichiometry of binding of enzyme to adenosine was determined as 1:0.16.
Inhibition by adenosine analogs. Phosphorylation of [3H]adenosine by L. donovani adenosine kinase is inhibited by tubercidin and 6-methylmer-
~- EAdo]pM I 0.2
To .oL O.O ,, '~
Direct demonstration of adenosine binding. Par-
~
]'~
[Mg'ATP]pM 3.0 q-
05
/ 0.3
/
2.5
. 12.5/
o.o2 004
/yy /
I/[A~o].~T j
B'O[
2.0
-7
< c
'- 3.0 y x
- 1.0 t°o
2.0 0.5 >~
~> 1.0 i
0.1 I/[ Mg-ATP]p~l
1
0.2
I
I
0.5 l/EAdenosine] p~1
I
1.0
Fig. 1. (A) Double reciprocal plots of initial velocity with variable ATP concentrations at several fixed concentrations of adenosine. Inset shows secondary plots of the slope (o--e) and the intercept (o--o) against inverse of adenosine concentration. (B) Double reciprocal plots of initial velocitywith variable adenosine concentration at several fixed concentrationsof ATP. Inset shows secondary plots of the slope (e--e) and the intercept (o--o) against inverse of [ATP-Mg2-] concentration.
184
I2] A ,
EAMP]pM..o
,_-;
'~'°~°1 ,
,
.0
oo/
/
i
B
~ [AMP]pM
:,I
/'
/
I Icz~
,0.8 E
~o/
le.E 0.6 n
~oo' 6o'
-6 0.4 E C
to 0 0.2 x ::> --,.,
I
I
I
I
I
I
0.1
0.2
0.5
0.4
0.01
0.02
I/EAdenosi ne -i IJ~1
I
0.05
1
0.04
I/[Mg-ATP] ~ I
Fig. 2. Double reciprocal plots of AMP inhibition with variable adenosine (A) and ATP (B) concentrations. Insets display slope ( e - - o ) and intercept (o--o) replots versus AMP concentration.
.
A
EMg- ADP-IPMjb
B
EMg-ADP] #M
1.2
%,
E
1.0
T~ •~ 0.8
52,0 Tu, E
~0~~
oo otO oo/
v
Aiw?
12_
-I.5
~: 0.6 -6 £ C
E cl
-I.0 c
O.4
to o
o x > 0.2
0.Sx
0.1
0.2
0.5
I/[Adenosine] ~1
0.4
0.02
0.04
0.04
>
0.08
I / [ M g - A T P ] 1.1[~I
Fig. 3. Double reciprocal plots of ADP inhibition with variable adenosine (A) and ATP (B) concentrations. Slope ( o - - e ) and intercept (o--o) replots are displayed against ADP concentration.
185 TABLE I Kinetic constants of adenosine and its analogs Kinetic constants ~
Adenosine/analogs
Adenosine Tubercidin 6-Methylmercaptopurine riboside
v,~x
Km
Vmax/Km
/':cat
K,,/gm
(Ixmol min 1 rag-<)
(IxM)
(1 min -l mg 1)
(s-l)
(s -1 txM l)
38.5 37 22.7
16 4.8 2.6
2.4 7.7 8.7
24 23 14
1.5 4.8 5.4
a Vm~xand Kr~ax determinations were done by spectrophotometric assay as described in Materials and Methods.
captopurine riboside [12]. Detailed studies indicate that both the analogs, besides being substrates themselves, behave as competitive inhibitors of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. The K i (slope) for tubercidin and 6-methylmercaptopurine riboside were determined as 70 IxM. Table I depicts composite kinetic constants of these compounds computed from spectrophotometric assay. Discussion
The results of the kinetic studies and equilibrium dialysis experiment are consistent with an ordered Bi-Bi addition of substrate and release of products with adenosine being the first substrate to bind and AMP the last product to be released. Schematically it may be shown as in Fig. 4. Our studies are in agreement with the kinetic properties of the enzyme from human placenta [26] but differ from the results obtained with enzymes from Ehrlich ascites tumor [27] and murine leukemia L1210 cell [28] lines with regard to order and sequence of addition of substrates. The kinetic properties and values for the kinetic constants of L. donovani adenosine kinase may be related to in vivo regulation of the enzyme. The phenomenon of adenosine kinase in-
Ad
ATP
1 E~--~E
ADP
T
Ad _
Ad ~ c-AMP EATP .~-----F_ADP ~
AMP
".c-AMP F
[
"E
Fig. 4. Reaction mechanism for L. donovani adenosine kinase.
hibition by AMP and ADP is interesting for several reasons. K~ values for AMP and ADP are in the range of their intracellular concentrations [29]. These inhibitions are consistent with the concept of regulation by ATP 'charge' as proposed by Atkinson and Fall [30]. The hypothesis proposed by them provides for ATP conservation within the cell so that the rate of reactions utilizing ATP is decreased when the ratio of [ATP]/[AMP] +[ADP] becomes low. Concentration of ATP has been shown to be about 50 times greater in mouse L-cells than in L. donovani [31]. Thus, the possibility of a correlation between the level of ATP and phosphorylating ability of adenosine kinase within L. donovani cells is not unlikely. Regarding the inhibitor and substrate specificity of L. donovani adenosine kinase it is interesting to note that both tubercidin and 6-methylmercaptopurine riboside are inhibitors of adenosine phosphorylation in vitro and parasite multiplication as well [8,19]. The studies indicate that at 50 txM substrate concentrations of each, reaction rates are in the order tubercidin> adenosine> 6-methylmercaptopurine riboside [12] whereas determination of Vma×/Km and Kcat/K m values fall in the sequence 6-methylmercaptopurine riboside>tubercidin>adenosine. This observation, together with other reports on the intracellular concentration of adenosine in eukaryotic cells (1 txM) [32,33], indicate that the efficacy of the compounds in vivo would be determined by the intracellular pool of adenosine. The results also suggest that in the absence of phosphotransferase activity in L. donovani [9] phosphorylation of nucleosides and their analogs is carried out mostly, if not entirely, by adenosine kinase. Furthermore, the observation that adenosine can be directly metabolized through three
186
different pathways in L. donovani, namely, phosphorylation, deamination and phosphorolysis [9], indicates that the K m value for adenosine with respect to adenosine kinase, adenosine deaminase and adenosine phosphorylase should determine which pathway will be favored in the presence of the substrate analogs. Thus the existence of multiple purine salvage pathways in L. donovani [3] indicates that perhaps in no case would inhibition of adenosine kinase alone be lethal unless other enzymes of salvage pathway are selectively targeted. Nevertheless, the observation reported
here could be useful while studying the role of this enzyme during parasite multiplication. Acknowledgements The authors wish to thank Mr. H.N. Dutta and his associates for art work. Thanks are also due to Drs. A.N. Bhaduri and Samit Adhya of this Institute for careful reading of the manuscript. D.B. is supported by a Junior Research Fellowship from the indian Council of Medical Research, New Delhi, India.
References 1 Gutteridge, W.E. and Coombs, G.H. (1977) Biochemistry of Parasitic Protozoa, University Park Press, Baltimore. 2 Walsh, C.J. and Sherman, I.W. (1968) Purine and pyrimidine synthesis by the avian malarial parasite Plasmodium lophurae. J. Protozool. 15,763-770. 3 Marr, J.J., Berens, R.L. and Nelson, D.J. (1978) Purine metabolism in Leishmania donovani and Leishmania braziliensis. Biochim. Biophys. Acta 544, 360-371. 4 Ceron, C.R., Caldas, R.A., Felix, C.F., Mundim, M.H. and Roitman, I. (1979) Purine metabolism in trypanosomatids. J. Protozool. 26, 479-483. 5 K6nigk, E. and Putfarkan, B. (1980) Stage-specific differences of a perhaps signal-transferring system in Leishmania donovani. Tropenmed. Parasitol. 31,421-424. 6 K6nigk, E. (1978) Purine nucleotide metabolism in promastigotes of Leishmania tropica: inhibitory effect of allopurinol and analogues of purine nucleosides. Tropenmed. Parasitol. 29, 435-438. 7 Lafon, S.W., Nelson, D.S., Berens, R.L. and Mart, J.J. (1982) Purine and pyrimidine salvage pathways in Leishmania donovani. Biochem. Pharmacol. 31,231-238. 8 Iovannisci, D.M. and Ullman, B. (1984) Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity. Mol. Biochem. Parasitol. 12, 139 151. 9 Looker, D.L., Berens, R.L. and Mart, J.J. (1983) Purine metabolism in Leishmania donovani amastigotes and promastigotes. Mol. Biochem. Parasitol. 9, 15 28. 10 Iovannisci, D.M., Kaur, K., Young, L. and Ullman, B. (1984) Genetic analysis of nucleoside transport in Leishmania donovani. Mol. Cell. Biol. 4, 1013-1019. 11 Aronow, B., Kaur, K., McCartan, K. and Ulhnan, B. (1987) Two high affinity nucleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22, 29-37. 12 Datta, A.K., Bhaumik, D. and Chatterjee, R. (1987) Isolation and characterization of adenosine kinase from Leishmania donovani. J. Biol. Chem. 262, 5515-5521. 13 Lindberg, B., Klenow, H. and Hansen, K. (1967) Some properties of partially purified mammalian adenosine kinase. J. Biol. Chem. 242, 350~356. 14 Divekar, A.Y. and Hakala, M.T. (1971) Adenosine kinase of sarcoma 180 cells (N~'-substituted adenosines as
substrates and inhibitors). Mol. Pharmacol. 7,663-673. 15 Gudas, L.J., Cohen, A., Ullman, B. and Martin, D.W. Jr. (1978) Analysis of adenosine mediated pyrimidine starvation using cultured wild type and mutant mouse T-lymphoma cells. Somatic Cell Genet. 4,201 219. 16 Divekar, A.Y., Kenny, L.N. and Hakala, M.T. (1971) Changes in Sarcoma-180 (S-180) cells associated with resistance to adenosine (AR) analogs. Fed. Proc. 30, 1253. 17 Bennett, L.L., Jr., Brockman, R.W., Schnebli, H.P., Chumley, S., Dixon, G.J., Schabel, F.H., Skipper, H.E., Montgomery, J.A. and Thomas, J. (1965) Activity and mechanism of action of 6-methylthiopurine riboside in cancer cells resistant to 6-mercaptopurine. Nature 205, 1276-1279. 18 Ho, D,H.W.. Luce, J.K. and Frei, E., [lI (1968) Distribution of purine ribonucleoside kinase and selective toxicity of 6-methylpurine ribonucleoside. Biochem. Pharmacol. 17, 1025-1035. 19 Acs, G., Reich, E. and Mori, M. (1964) Biological and biochemical properties of the analogue antibiotic tubercidin. Proc. Natl. Acad. Sci. USA 52,493-501. 20 Schwartzman, J.D. and Pferferkorn, E.R. (1982) Toxoplasma gondii purine synthesis and salvage in mutant host cells and parasites. Exp. Parasitol. 53, 77-86. 21 Dovcy, H.F., Mckerrow, J.H, and Wang, C.C. (1985) Action of tubercidin and other adenosine analogs in Schistosorna mansoni schistosomules. Mol. Biochem. Parasitol. 16, 185 198. 22 Berens, R.L. and Marr, J.J. (1986) Adenosine analog metabolism in Giardia lamblia. Biochem. Pharmacol. 35, 4191-4197. 23 Ghosh, A.K., Bhattacharyya, F.K. and Ghosh, D.K. (1985) Leishmania donovani amastigote inhibition and mode of action of berberine. Exp. Parasitol. 60,404-413. 24 Cleland, W.W. (1970) Steady state kinetics. In: The Enzymes (Boyer, P.D., ed.) 3rd edn. w)l. 2, pp. 1-65, Academic Press, New York. 25 Cleland, W.W. (1963) The kinetics of enzyme catalysed reactions with two or more substrates or products. Biochim. Biophys. Acta 67, 104-137. 26 Palella, T.D., Andres, C.M. and Fox, I.H. (1980) Human
187
27
28
29 30
placental adenosine kinase (kinetic mechanism and inhibition). J. Biol. Chem. 255, 5264-5269. Henderson, J.F., Mikoshiba, A., Chu, S.Y. and Caldwall, I.C. (1972) Kinetic studies of adenosine kinase from Ehrlich ascites tumor cells. J. Biol. Chem. 247, 1972-1975. Chang, C-H, Cha, S., Brockman, R.W. and Bennett, L.L. Jr. (1983) Kinetic studies of adenosine kinase from L1210 cells: A model enzyme with a two-site ping-pong mechanism. Biochemistry 22, 600-611. Bartlett, G.R. (1968) Phosphorous compounds in the human erythrocyte. Biochim. Biophys. Acta 156, 221-230. Atkinson, D.E. and Fall, I. (1967) Adenosine triphosphate conservation in biosynthetic regulation. J. Biol.
Chem. 242, 3241-3242. 31 Lafon, S.W., Nelson, D.J., Berens, R.L. and Mart, J.J. (1985) Inosine analogs, their metabolism in mouse L cells and in Leishmania donovani. J. Biol. Chem. 260, 9660-9665. 32 Fox, A.C., Reed, G.E., Glassman, E., Kaltman, A.J. and Silk, B.B. (1974) Release of adenosine from human hearts during angina induced by rapid atrial pacing. J. Clin. Invest. 53, 1447-1457. 33 Mills, G.C., Schmalsteig, F.C., Trimmer, K.B., Goldman, A.S. and Goldblum, R.M. (1976) Purine metabolism in an adenosine deaminase deficiency. Proc. Natl. Acad. Sci. USA 73, 2867-2871.