Phenylglyoxal inactivation of the mitochondrial adenosine triphosphatase from Trypanosoma cruzi

Phenylglyoxal inactivation of the mitochondrial adenosine triphosphatase from Trypanosoma cruzi

Molecular and Biochemical Parasitology, 5 ( 1982 ) 371 - 379 Elsevier Biomedical Press 371 PHENYLGLYOXAL INACTIVATION OF THE M1TOCHONDRIAL ADENOSINE...

443KB Sizes 3 Downloads 78 Views

Molecular and Biochemical Parasitology, 5 ( 1982 ) 371 - 379 Elsevier Biomedical Press

371

PHENYLGLYOXAL INACTIVATION OF THE M1TOCHONDRIAL ADENOSINE TRIPHOSPHATASE FROM T R Y P A N O S O M A CR U Z I

MARIA ANTONIA CATALDI DE FLOMBAUMand ANDRES O.M. STOPPANI Centro de lnvestigaciones Bioenerg~ticas y Instituto de Quimica Biol6gica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina

(Received 25 January 1982; accepted 23 February 1982)

The reaction of Trypanosoma cruzi Mg2+-stimulatedadenosine triphosphatase (ATPase, coupling factor 1, or F t ) with phenylglyoxal, a dicarbonylic compound, resulted in a rapid loss of its enzymatic activity. The inactivation showed pseudo-first-order kinetics with both membrane-bound and soluble F l -ATPase, the rate of the enzyme inactivation being faster in bicarbonate buffer (pH 7.9) than in borate buffer (pH 8.0). The log (pseudo-first-order rate constant) vs. log(phenylglyoxal concentration) plots obtained with the membrane-bound and soluble F~ -ATPase in bicarbonate buffer, and also with F i in borate buffer, had slopes of near 1.0 while the plot for the membrane-bound ATPase in borate buffer had a slope of 1.6. Second-order rate constants (in mM-1 • min-t) were 55 (for both ATPase preparations in bicarbonate buffer) and 34 (for the membrane-bound ATPase in borate buffer). When the reaction was performed in the presence of ATP, the rate of inactivation was significantly decreased. It is concluded that, as in the mammalian F~ -ATPase, arginyl residues play an essential role in T. cruzi mitochondrial ATPase, probably at the hydrolytic site. Key words: Trypanosoma cruzi, Mitochondrial adenosine triphosphatase, Coupling factor Ft, Arginyl residues, Phenylglyoxal.

INTRODUCTION The membrane-associated, Mg2+-stimulated adenosine triphosphatase (ATPase, EC 3.6.1.3; coupling factor 1, or F1) is involved in the synthesis of ATP during the terminal steps of oxidative phosphorylation. In contrast to extensive studies performed on mammalian, yeast and bacterial ATPases, the information available on the protozoan enzyme is relatively limited. Mitochondrial (kinetoplast) ATPase was demonstrated in T r y p a n o s o m a cruzi (the agent of Chagas' disease) by Sastre and Stoppani [1 ] and Frasch et al. [2]. Frasch et al. [3] were able to isolate T. cruzi mitochondrial ATPase by chloroform extraction, and Cataldi de Flombaum et al. [4] pu.rified the soluble enzyme and established some of its properties [4, 5]. One approach to gain insight into an enzyme mechanism is through chemical modification of particular residues by means of group-specific reagents. Arginine residues play an essential role in many enzymes reacting with anionic substrates or ligands [6] 0166-6851/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress

372 and such a role of arginine residues has been established in various ATPases, including the mammalian mitochondrial ATPase [ 7 - 9 ] , the (Ca2++ Mg2÷)-ATPase from sarcoplasmic reticulum [10], the (K÷+ H÷)-ATPase [11] and the chloroplast ATPase [12]. In order specifically to modify arginyl residues in enzymes, phenylglyoxal, a dicarbonylic reagent, has been used extensively [6]. The reagent rapidly causes loss of activity, which can be directly correlated with the loss of a single arginyl residue per active site. In the present study we assayed phenylglyoxal on membrane-bound and soluble Fl from T. cruzi, keeping in mind that comparative studies of enzyme inhibition in parasitic protozoa may lead to the discovery of new chemotherapeutic agents. MATERIALS AND METHODS

T. cruzi. Tulahuen strain was grown at 28°C in Warren's [13] liquid medium and the cultured epimastigotes were collected as described in [3].

ATPase preparation. The membrane-bound enzyme was prepared as described [3]. The fraction obtained after 30 min centrifugation at 32 000 × g was suspended in 14 mM sucrose, 2.5 mM KCI, 2 mM EDTA, pH 7.0. F1 was isolated and purified as described by Cataldi de Flombaum et al. [4]. The specific activity of the membrane-bound and the soluble F~-ATPase preparations (units/mg of protein) was 0.3 and 2 5 - 2 9 , respectively (one unit of ATPase activity is the amount of enzyme which hydrolyzes 1 #mol of ATP per min).

Modification o f A TPase with phenylglyoxal. These experiments were performed at 30°C by addition of reagent to the enzyme solution (duplicate samples). With the membranebound ATPase, protein concentration was within the range of 2 . 6 - 6 . 0 mg/ml, in 0 . 4 0.5 ml 90 mM borate (Na; pH 8.0) or 85 mM bicarbonate (Na; pH 7.9). Samples (40/A) of each incubation mixture were withdrawn at convenient time intervals and ATPase activity was measured. With the soluble F~, protein concentration was 10 gg/ml, other experimental conditions being as described above. Controls in the absence of inhibitor were similarly treated. Phenylglyoxal was dissolved in the indicated buffer just before use and its concentration was determined by absorbance at 253 nm using an extinction coefficient of 12.6 mM -~ - cm -~ [14]. Protection experiments were performed as described above except that the enzyme was preincubated with ATP for 5 rain before adding phenylglyoxal. Parallel controls of non-enzymic hydrolysis of ATP were also included and used to correct the experimental rates of hydrolysis. Each experiment was repeated at least three times, with consistent results. The figures represent the result of a typical experiment. The kinetics of ATPase inactivation were examined with semilogarithmic plots of the ratio of inhibited to original enzyme activity as a function of time of enzyme incubation with phenylglyoxal. These plots allowed one to determine the time for 50 per cent inactivation (To.s) for each phenylglyoxal concentration. From these values, the

373 pseudo-first-order rate constant kob s was calculated. Representation o f kob s as a function o f phenylglyoxal concentration permitted calculation o f the second-order rate constant for the ATPase-inactivation reaction. Finally, the reaction order with respect to phenylglyoxal was determined from a plot o f log kob s against log(phenylglyoxal concentration) [8, 15]. In this type o f representation, a straight line should be obtained with a slope equal to n, n being the number o f molecules o f inhibitor reacting with each active site to produce an inactive complex.

Assay of ATPase activity ATPase activity was measured at 30°C in an incubation mixture (final volume, 1 ml) containing 150 mM Tris-HC1 (pH 7.6), 3 mM ATP, 4 mM MgC12 and 0.25 mM EGTA. The reaction was started by adding the enzyme as stated in each case. After 20 min, 0.1 ml o f 50% (w/v) trichloroacetic acid was added. Orthophosphate concentration was determined b y the colorimetric method o f Fiske and Subbarow [16].

Protein determination. Protein concentration o f the soluble and the membrane-bound ATPase preparation was measured using the methods of Bensadoun et al. [17] and Lowry et al. [18] respectively, bovine serum albumin being used as standard protein.

Chemicals. Phenylglyoxal, ATP and bovine serum albumin were obtained from Sigma Chemical Co., St. Louis, MO. All other reagents were of analytical grade. RESULTS

Inactivation of membrane-bound F 1-A TPase. Incubation o f T. cruzi membrane-bound ATPase with phenylglyoxal caused rapid inactivation o f the enzyme (Fig. 1). The rate 100

,

,

,

,

,

,

80

>-

60

F- 40

So

2O

i

0

i

5

i

i

i

10 "rIME ( m i n )

i

15

i

20

Fig. 1. Inactivation of membrane-bound ATPase by phenylglyoxal in bicarbonate (Bi) or borate (Bo) buffer solutions. The reaction mixture contained the ATPase preparation (2.5 (Bi) or 5.0 (Bo) mg of enzyme protein/ml); 6 mM phenylglyoxal; 85 mM bicarbonate (Na; pH 7.9) or 90 mM borate (Na; pH 8.0). After incubation for the time indicated on the abscissa, samples (40 t~l) were withdrawn and ATPase activity was measured. Other experimental conditions were as described under Materials and Methods. The points represent duplicate measurements; the deviation from the mean was less than 5%.

374

o f inactivation depended on the buffer anion, as shown b y comparing the decline o f activity in bicarbonate and bozate buffers. In good agreement with the effect of bicarbonate on the reaction o f phenylglyoxal with arginine [19] the rate o f enzyme inactivation was faster when using bicarbonate buffer. The semilogarithmic plots in Fig. 2A show the enzyme inactivation at various phenylglyoxal concentrations in bicarbonate buffer. The inactivation rates were dependent on the phenylglyoxal concentration and obeyed pseudo-first-order kinetics with respect to enzyme active sites, until 90% enzyme inactivation. When the inactivation rate constants, calculated from the primary plots, were plotted as log kob s against log(phenylglyoxal concentration) (Fig. 2A, inset), a linear relationship was obtained. According to Scrutton and Utter [20], the rate constant kob s could be expressed b y equation (1) kobs = k [PG] n

(1)

where k is a proportionality constant, [PG] is phenylglyoxal concentration and n is the order o f reaction with respect to phenylglyoxal. Since log kobs = log k + n log [PG], n was obtained from the slopes o f the log/log plot in Fig. 2A (inset). With the membrane-bound ATPase in bicarbonate buffer, a slope o f 1.0 was obtained, indicating the reaction o f one phenylglyoxal molecule with 1 arginyl residue per active site. Fig. 2B shows the results of a similar experiment using borate instead o f bicarbonate buffer.

100

100,

80

80

60

60

~ 40

A40 o P.

20

20 A

-0.4

0

0

0.4

Log[PG](m M) 5

10 TIME (rain)

15

0

I 5

I 10 TIME

0.5 1.0 Log[P0]( mM)

1'5

2;0

(mini

Fig. 2. Kinetics of the inactivation of membrane-bound ATPase by phenylglyoxal. (A) The reaction mixture contained 2.5 mg/ml of enzyme protein, 85 mM bicarbonate (pH 7.9) and phenyiglyoxal (mM) as indicated by the figures near the straight lines. Inset: determination of the order of the reaction between ATPase and phenylglyoxal with respect to phenylglyoxal (PG); kobs(S) values were calculated from the semilogarithmic plots. (B) The reaction mixture contained 5 mg/ml of enzyme protein, 90 mM borate (pH 7.9) and phenylglyoxal (raM) as indicated by the figures. After incubation for the time indicated on the abscissa, samples were taken and ATPase activity was measured. Inset: as in Fig. A. Other conditions were as described in Fig. 1 and under Materials and Methods. F o and F, original and residual (phenylglyoxal-inhibited) ATPase activity, respectively.

375 After 5 min incubation and until 70% of the active sites had been inactivated, phenylglyoxal inhibition again exhibited pseudo-first-order kinetics with respect to enzyme, but at variance with the results using bicarbonate buffer, the n value in borate buffer was 1.6 (Fig. 2B, inset).

Inactivation of soluble F1-A TPase. Preliminary experiments demonstrated that borate buffer affected F1 stability. Thus, when the enzyme preparation was incubated in 85 mM borate (pH 8.0) at 30°C, 30% o f the initial activity was lost after 5 min, and 50% after 1 h incubation. At variance with these results, incubation of F1 in 90 mM bicarbonate (pH 7.9), for 1 h, did not affect the enzyme activity and, accordingly, the bicarbonate medium was adopted for the study of phenylglyoxal effect. In good agreement with the results obtained with the membrane-bound enzyme (Fig. 2A), the semilogarithmic plots in Fig. 3 show that the reaction was pseudo-first-order with respect to the enzyme active sites and the log/log plot in the inset gives an n value equal to 0.9. Fig. 4 shows the representation of kob s values as a function o f phenylglyoxal concentration, for both the soluble and the membrane-bound F1. The straight lines obtained passed through the origin, thereby indicating that there was no enzyme-reagent complex formed prior to inactivation. From the slopes o f the lines, the second.order-rate constants were calculated, the values obtained (in mM • min -1 ) being 55 for the two ATPase preparations in bicarbonate buffer, and 34 for the membrane-bound ATPase in borate buffer. Interestingly, a value o f 22 was reported for mammalian membrane-bound F~, but the constant value for the soluble Fa -ATPase was only about 2.7 [9].

100

40

i

i

r

I

q

80

o

60

30

T

c_.

Oi

A

.~ 4o 20

.o #

So

i

o 10

20 0

0.4

O.8

Log[PG](mH)

2 0

5

10 TIME { m i n )

15

20

4

6

[PHENYL6LYOXAL] ( mM )

Fig. 3. Kinetics of the inactivation of soluble F 1 by phenylglyoxal. The incubation system contained FI preparation (10 tag of enzyme protein/ml), 85 mM bicarbonate (pH 7.9) and phenylglyoxal (mM) as indicated by the figures near the straight lines. Other conditions were as described in Fig. 2. Fig. 4. Effect of phenylglyoxal concentration on the pseudo-first-order rate constant (kobs) of ATPase inactivation. The ko~ values were calculated from data in Fig. 2 and 3. o, membrane bound ATPase in bicarbonate buffer; o, same, in borate buffer; e, soluble F~ -ATPase in bicarbonate buffer. Bi and Bo as in Fig. 1.

376

Effect of ATP. In accordance with observations by Marcus et al. [8] with beef-heart mitochondrial ATPase, the inactivation of the T. cruzi enzyme by phenylglyoxal was retarded by ATP. Fig. 5 shows the effect of ATP on the kinetics of phenylglyoxal inactivation of soluble F~ and Table I shows the diminution ofkob s in the presence of ATP, with both the soluble and the membrane-bound enzyme.

100 ~

,

,

,

80 60 18 .--t 4O

>o 2O

0

I'O

;

1'5

20

TIME (min)

Fig. 5. Effect of ATP on phenylglyoxal inactivation of soluble F 1-ATPase. The reaction mixture contained F~ preparation (10 vg of enzyme protein/ml); 3 mM phenylglyoxal and 85 mM bicarbonate (pH 7.9). The figures near the straight lines indicate ATP concentration (mM). Other experimental conditions were as described under Materials and Methods.

TABLE I Effect of ATP on the pseudo-first-order rate constant of phenylglyoxal inactivation of T. cruzi ATPase ATPase preparation

Phenylglyoxal (mM)

ATP (mM)

kobs (min- 1)

Decrease of ko~ (%)

Soluble FI

3

0 9 18

0.18 0.08 0.06

55 66

0 10 20

0.38 0.06 0.04

78 84

Membrane-bound

6

Incubation in 85 mM bicarbonate buffer (pH 7.9). Other experimental conditions were as described in Fig. 5 and under Materials and Methods.

377 DISCUSSION Although phenylglyoxal seems to be less specific than butanedione as a reagent for arginyl residues in proteins [6, 8], it has been extensively used as a chemical modifier for enzymes that exhibit butanedione-sensitive arginyl residues. Phenylglyoxal interacts with arginyl residues to give a product which, depending on the experimental conditions, contains either one or two-phenylglyoxal moieties per group [6]. This product is relatively stable and dissociates to regenerate arginine only on prolonged incubation. The stability of the product is helpful for the identification of the modified arginyl residue in the primary structure. An important side reaction of phenylglyoxal is rapid Schiff base formation with a-amino groups of peptides and e-amino groups of lysine; in these cases a stoichiometry of greater than 2 can be expected [6]. Accordingly, the fractional reaction order in Fig. 2B could imply the binding of more than one molecule of phenylglyoxal to the enzyme. As suggested by Scrutton and Utter [20] for the case of avididin inhibition of pyruvate carboxylase, statistical distribution among the number of molecules bound per active site could give the observed 1.6 value. Since fractional reaction order was observed only when using borate, the possibility that borate might facilitate a conformational change making a second arginyl residue accessible to phenylglyoxal [9] should be also considered. The occurrence of such a change is suggested by the inactivating effect of borate on soluble T. cruzi F1. Although direct evidence for the modification of arginyl residues is lacking, the resuits reported here are consistent with the view that phenylglyoxal modifies one arginyl residue at, or near the active site of T. cruzi, soluble or membrane-bound F1-ATPase. The protective effect of ATP (Fig. 5 and Table I) is evidence in support of the function of one arginyl residue at the ATPase hydrolytic site. Concerning the buffer effect in Figs. 1 - 3 , it is worth noting that the rate of reaction of phenylglyoxal with arginine and arginyl residues [19] is faster in bicarbonate than in other buffers, due to complex formation between bicarbonate and the guanidinium group, which lowers the pK a and thus promotes nucleophilic attack by the latter group at the carbonyl carbon of phenylglyoxal [6]. Conversley, the reaction of arginine and a-oxo aldehydes (such as phenylglyoxal) is inhibited by borate [21]. Since the stoichiometry of phenylglyoxal inactivation of the soluble and the membrane-bound F1 in bicarbonate buffer were similar, it must be inferred that with the membrane-bound enzyme the binding site of the reagent causing inhibition of activity was at the F1 hydrolytic site. The kinetics of phenylglyoxal inhibition of T. cruzi ATPase does not show significant differences in the reactivity of the parasite (reported here) and mammalian host (as reported in the literature) enzymes, thus making unlikely the inhibition of T. cruzi ATPase by dicarbonylic compounds the basis for chemotherapy of Chagas' disease. ACKNOWLEDGEMENTS This work was aided by grants from Secretaria de Estado de Ciencia y Tecnologia

378

( A r g e n t i n a ) , t h e Scientific Office o f t h e O r g a n i z a t i o n o f A m e r i c a n States a n d t h e U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for R e s e a r c h a n d T r a i n i n g i n T r o p i c a l Diseases. B o t h a u t h o r s are C a r e e r I n v e s t i g a t o r s C o n s e j o N a c i o n a l de I n v e s t i g a c i o n e s Ci6ntificas y T6cnicas, R e p f b l i c a A r g e n t i n a . REFERENCES 1

Sastre, M.B.R. de and Stoppani, A.O.M. (1973) Demonstration of a Mg2÷-activated adenosine triphosphatase in Trypanosoma cruzi. FEBS Lett. 3 1 , 1 3 7 - 1 4 2 . 2 Frasch, A.C.C., Segura, B.L., Cazzulo, ,J.J. and Stoppani, A.O.M. (1978) Adenosine triphosphatase activities in Trypanosoma cruzi. Comp. Biochem. Physiol. 60B, 2 7 1 - 2 7 5 . 3 Frasch, A.C.C., Cazzulo, J.J. and Stoppani, A.O.M. (1978) Solubilization and some properties of the Mg2÷-activated adenosine triphosphatase from Trypanosoma cruzi. Comp. Biochem. Physiol. 61B, 2 0 7 - 2 1 7 . 4 Cataldi de Flombaum, M.A., Frasch, A.C.C. and Stoppani, A.O.M. (1980) Adenosine triphosphatase from Trypanosoma cruzi: purification and properties. Comp. Biochem. Physiol. 65B, 103-109. 5 Cataldi de Flombaum, M.A. and Stoppani, A.O.M. (1981) Influence of efrapeptin, aurovertin and citreoviridin on the mitochondrial adenosine triphosphatase from Trypanosoma cruzi. Mol. Biochem. Parasitol. 3 , 1 4 3 - 1 5 5 . 6 Riordan, J.F. (1979) Arginyl residues and anion binding sites in proteins. Mol. Cell Biochem. 26, 7 1 - 9 2 . 7 Ferguson, S.J., Lloyd, W.J. and Radda, G.K. (1974) An unusual and reversible chemical modification of soluble beef-heart mitochondrial ATPase. FEBS Lett. 3 8 , 2 3 4 - 2 3 6 . 8 Marcus, F., Shuster, S.M. and Lardy, H.A. (1976) Essential arginyl residues in mitochondrial adenosine triphosphatase. J. Biol. Chem. 251, 1775-1780. 9 Frigeri, L., Galante, Y.M., Hanstein, W.G. and Hatefi, Y. (1977) Effect of arginine binding reagents on ATPase and ATP-P i exchange activities of mitochondrial ATP synthetase complex (complex V). J. Biol. Chem. 252, 3 1 4 7 - 3 1 5 2 . 10 Murphy, A.J. (1976) Arginyl residue modification of the sarcoplasmic reticulum ATPase protein. Biochem. Biophys. Res. Commun. 70, 1048-1054. 11 Schrijen, J.J., Luyben, W.A.H.M., De Pont, J.J.H.H.M. and Bonting, S.L. (1980) Studies on (K÷+ H÷)-ATPase. I. Essential arginine residue in its substrate binding center. Biochim. Biophys. Acta 2 9 7 , 3 3 1 - 3 4 4 . 12 Andreo, C.S. and Vallejos, R.H. (1977) An essential arginyl residue in the soluble chloroplast ATPase. FEBS Lett. 7 8 , 2 0 7 - 2 1 0 . 13 Warren, L. (1960) Metabolism of Schizotrypanum cruzi Chagas. I. Effect of culture age and substrate concentration on respiratory rate. J. Parasitol. 46, 5 2 9 - 5 3 9 . 14 Kohlbrenner, W.E. and Cross, R.L. (1978) Efrapeptin prevents modification by phenylglyoxal of an essential arginyl residue in mitochondrial adenosine triphosphatase. J. Biol. Chem. 253, 7609-7611. 15 Wong, S.S. and Wong, L.-J.C. (1981) Evidence for an essential arginine residue at the active site ofEscherichia coli acetate kinase. Biochim. Biophys. Acta 660, 141-147. 16 Fiske, C.H. and Subbarow, Y. (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 6 6 , 3 7 5 - 4 0 0 . 17 Bensadoun, A. and Weinstein, D. (1976) Assay of protein in the presence of interfering materials. Analt. Biochem. 7 0 , 2 4 1 - 2 5 0 . 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1 9 3 , 2 6 5 - 2 7 5 .

379

19 20 21

Cheung, S.-T. and Fonda, M.L. (1979) Reaction of phenylglyoxal with arginine. The effect of buffers and pH. Biochem. Biophys. Res. Commun. 9 0 , 9 4 0 - 9 4 7 . Scrutton, M.C. and Utter, M. (1965) Pyruvate carboxylase. V Interaction of the enzyme with adenosine triphosphate. J. Biol. Chem. 240, 3714-3723. Rogers, T.B., BC~rrensen, T. and Feeney, R.E. (1978) Chemical modification of the arginines in transferrins. Biochemistry 17, 1105-1109.