Mechanism-based inhibition of proline dehydrogenase by proline analogues

Mechanism-based inhibition of proline dehydrogenase by proline analogues

Biochimica et Biophysica Acta, 1202 (1993) 77-81 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00 77 BBAPRO 34544 Me...

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Biochimica et Biophysica Acta, 1202 (1993) 77-81 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

77

BBAPRO 34544

Mechanism-based inhibition of proline dehydrogenase by proline analogues Denis Tritsch, Hiba Mawlawi and Jean-Francois Biellmann Laboratoire de Chimie Organique Biologique, URA CNRS 31, Facult~ de Chimie, UniversitdLouis Pasteur, Strasbourg (France)

(Received 12 January 1993)

Key words: Proline dehydrogenase; Enzyme inhibition; Mechanism-based inhibition; Mitochondrion; (G. morsitans); (Rat liver) The inactivation of proline dehydrogenase by several L-Pro analogues was investigated with the aim to block the essential metabolic pathway of tsetse flies allowing the degradation of L-Pro to L-Glu. In vitro studies on rat liver mitochondria showed that only 4-methylene-L-proline was able to inactivate proline dehydrogenase. The inactivation kinetics agreed with a mechanism-based inhibition. The other tested analogues E- and Z-4-fluoromethylene-L-proline, and c/s and trans-5-ethynyl-D,L-proline were neither substrate nor inactivator of the enzyme. In vivo 4-methylene-L-proline showed no toxicity against Drosophila flies, but was lethal for Glossina pallidipes flies. This result allows the consideration of 4-methylene-L-proline as an attractive compound molecule in the struggle against tsetse flies.

Introduction Proline dehydrogenase (EC 1.5.99.8), commonly referred to as proline oxidase, is the first enzyme of the catabolic pathway of L-Pro to L-Glu [1,2]. The enzyme oxidises L-Pro (1 in Scheme I to Al-pyrroline-5-carbo xylic acid (2 in Scheme I). The latter, in equilibrium with its open form glutamic-y-semialdehyde, is oxidised to L-Glu by a NAD+-dependent AX-pyrroline-5 carboxylate dehydrogenase. Proline dehydrogenase has been found in mammals, insects, plants and bacteria. In animal tissue, proline dehydrogenase is located in the inner membrane of mitochondria [3] where it is coupled to the respiratory chain. Ubiquinone acts as the first electron acceptor [4]. Proline dehydrogenase from rat liver mitochondria was solubilised with Triton X-100 [5] or snake venom [6]. No earlier reports of the purification of proline dehydrogenase from animal tis-

~ C O O H

=HN~,.,~COO

I

H

+ H ÷ + 2e

I

H

H 2

Scheme I.

Correspondence to: J.-F. BieUmann, Laboratoire de Chimie Organique Biologique, Facult6 de Chimie, Universit6 Louis Pasteur, 1 rue Blaise Pascal, 67008 Strasbourg Cedex, France.

sues have been found. The enzyme from Escherichia coli K12 cytoplasmic membrane has been shown to be a dimeric flavoprotein of 260 kDa [7]. Some insects use L-Pro as an energy source during flight [8]. A high concentration of L-Pro has been detected in the flight muscle of insects such as Locusta migratoria [9,10], Leptinotarsa decemlineata [11,12], Phormia regina [13] and most interestingly the tsetse fly Glossina morsitans [14]. In the tsetse fly, L-Pro is the sole energy source during flight [14-16]. Inhibition of proline dehydrogenase may, therefore, provide a strategy in controlling the tsetse fly. Hitherto, proline dehydrogenase activity in insects has been reported, but no details on the properties of the enzyme have been given. Some inhibitors for proline dehydrogenase have been mentioned in the literature. Inhibitors of the respiratory chain are non-specific inhibitors which abolish proline dehydrogenase activity by preventing its regeneration [2]. The proline analogue L-3,4-dehydroproline [17], D- and L-lactic acid, and pyruvic acid are known to be competitive inhibitors [7,18]. Pyrrole-2carboxylic acid is a non-competitive inhibitor for proline dehydrogenase activity from rat liver [18]. We found that L-pyroglutamic acid and 2-imidazolidone-4carboxylic acid were mixed-type inhibitors (Tritsch, D., unpublished results). L-Thiazolidine-4-carboxylic acid inhibits proline oxidation in barley mitochondria [19]. In order to explore the inhibition of proline dehydrogenase, the following proline analogues in which a double or triple bond was introduced into the

78 F

~

H

C~H

i

C~H

I

H 3

I

H 4

~COOH HC~C"

~N /

~H

H 5 ~ C O O H

Hc~C'"" ~ N f

I It

I

H 7

6

SchemeII. proline skeleton were prepared: 4-methylene L-proline (3 in Scheme II, E- and Z-4-fluoromethylene-L-proline (4 and 5 in Scheme II), and c/s and trans-5-ethynylO,L-proline (6 and 7 in Scheme II) [20]. These analogues possess functions which could induce a mechanism-based inhibition. During the oxidation process, the substrate is converted to an electrophilic species susceptible to reaction with a nucleophilic group present in the active site. To test this assumption, we studied the influence of these proline analogues on the activity of proline dehydrogenase from rat liver. Due to the rapid loss of enzyme activity of the solubilized proline dehydrogenase, our attempts to purify the enzyme from rat liver mitochondria were unsuccessful (Mawlawi, H. and Tritsch, D., unpublished results). Thus, the inactivation studies were performed on mitochondria suspensions. Materials and Methods

Menadione, 2-aminobenzaldehyde and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride hydrate (INT) were purchased from Aldrich (Steinheim, Germany). L-Pro and bovine serum albumin were supplied by Sigma (St. Louis, MO, USA). The synthesis of the proline analogues (3-7 in Scheme II) has been described elsewhere [20]. Isolation of rat liver mitochondria. Mitochondria were isolated from Wistar rats as described [21]. The buffer was 0.25 M saccharose, 1 mM EDTA, 1 mM phosphate buffer (pH 7.4). The fractionated mitochondria were stored four days at -20°C before use [5]. Mitochondria were stored in small aliquots to avoid freeze-thawing operations which lead to large activity losses. The protein concentration, as determined by the modified Lowry method [22], was 37 mg/ml. Proline dehydrogenase assay. The activity of proline dehydrogenase was determined by a method derived from that described for the mitochondrial a-glycerophosphate dehydrogenase [23]. A menadione suspension was prepared prior to use by adding a 0.7% menadione solution in ethanol (0.3 ml) to 1.2 ml bovine

serum albumin solution (2 mg/ml) in 0.1 M phosphate buffer (pH 7.4). The assay was performed at 30°C in 0.1 M phosphate buffer (pH 7.4). The reaction was initiated by the addition of L-Pro (10 mM final concentration) to a test tube containing mitochondria suspension (1 mg protein/ml), potassium cyanide (1 mM), menadione suspension (35/zl) and INT (0.33 mM). The final volume was 1 ml. After a 2-min incubation with constant stirring, 0.4 ml of the reaction mixture was added to 0.4 ml trichloroacetic acid (10% in water) to stop the enzymatic reaction. The reduced dye was extracted twice with ethyl acetate (final volume 1.6 ml). The absorbance was measured at 490 nm (E = 20100 M - l c m -1) against an assay without proline on a Hewlett Packard HP 8451A spectrophotometer. The maximal rate and the Michaelis constant of L-Pro and 4-methylene-L-proline were determined as described above using concentrations of 2-10 mM. The proline analogues (4-7 in Scheme II) were tested for oxidation by proline dehydrogenase using concentrations up to 20 mM. The 2-aminobenzaldehyde method was also used to determine the enzymatic activity [24]. Inactivation of proline dehydrogenase. The mitochondria suspension (120/~1) was incubated at 30°C for 5 min in 0.1 M phosphate buffer (pH 7.4). The proline analogues (3-7 in Scheme II) were added to a final concentration of 20 mM (final volume 480 ~1). At various times, aliquots (80/zl) were withdrawn and the residual activity was determined as described above. In the case of (3), the inactivation rate (kobs) was determined for concentrations of 6-20 mM to calculate the maximal inactivation rate and the dissociation constant [25]. Measurements were made at least in duplicate.

Protection of proline dehydrogenase against inactivation. The inactivation experiments were carried out as above at a 20 mM concentration of 4-methylene-L-proline in the presence of L-Pro (10 mM) or potassium cyanide (2 mM) or D,L-lactate (25 mM) or reduced glutathione (1 mM). Irreversibility of the inactivation. Mitochondria suspension with inactivated proline dehydrogenase was centrifuged and the supernatant was discarded. The mitochondria pellet was washed twice with the phosphate buffer. The enzymatic activity was then determined after homogenization of the pellet. These operations were carried out at 4°C. The same process was carried out with the native mitochondria suspension. Results and Discussion

Oxydation of L-Pro and analogues 3-7 by proline dehydrogenase The assay is based on the reduction of 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride hydrate (INT) to the corresponding formazan

79 with menadione as electron transfer from proline dehydrogenase. Potassium cyanide was added to inhibit the respiratory chain, the natural regenerating system of the enzyme. The results were reproducible when formazan was carefully extracted. The velocity was constant, at least, up to 3 min of incubation time and proportional to protein concentrations < 1.2 mg/ml. Higher protein concentrations led to a poor extraction of the reduced dye. The apparent Michaelis constant of L-Pro was found to be 1.8 _+ 0.8 mM, close to the value using the same assay for the solubilised enzyme from rat liver ( K m = 2.3 mM) [18]. However, this was found to be lower than that obtained using the 2-amino benzaldehyde method (5 + 1 mM) [17]. Inhibition by substrate excess was observed for a L-Pro concentration above 10 mM in the tetrazolium/menadione assay and 25 mM in the 2-amino benzaldehyde assay. A maximal rate of 25 + 2/zmol reduced tetrazolium/min per g mitochondrial protein in the assay conditions was measured. Among the proline analogues, only 4-methylene-Lproline was oxidised by proline dehydrogenase at an appreciable rate, allowing an evaluation of the Michaelis constant (about 10 mM) and the maximal rate (about 17 + 2/zmol reduced INT/min per g mitochondrial protein). Because of the inactivation of the enzyme by 4-methylene-L-proline, only estimations of the Michaelis constant and of the maximal rate could be made. The presence of a methylene group at position 4 does not seem to greatly affect the recognition and the oxidation rate. 4-Hydroxy-L-Pro was shown to be oxidised by mitochondria [26,27], but a separate enzyme seems to be available [27]. The two isomers of 4-fluoromethylene-L-proline were poor substrates of the enzyme. The oxidation rate, at a concentration of 10 mM was 1.7/zmol reduced INT/min per g mitochondrial protein. No reduction of INT was observed with compounds 6 and 7. They were not oxidised by proline dehydrogenase. However, we have shown that mitochondria catalyse the transformation of compounds 6 and 7 to products which reacted with 2aminobenzaldehyde (Am~x -- 436 nm). They could result from the oxidation of the D isomers to the corresponding Al-pyrroline-2-carboxylic acid derivatives by Damino-acid oxidase, also present in mitochondria suspension [17]. Indeed, D-Pro is oxidised by mitochondria suspension to Al-pyrroline-2-carboxylic acid, which reacts with 2-aminobenzaldehyde [28] to give a complex with a hm~, of 436 nm. Proline dehydrogenase was not inactivated by compounds 4-7. So, the absence of INT reduction was not due to a rapid loss of activity.

Inactivation of proline dehydrogenase by 4-methylene-Lproline 4-Methylene-L-proline inactivated the enzyme with pseudo-first-order kinetics. Saturating kinetics were

observed, indicating the formation of a complex between enzyme and 4-methylene-L-proline prior to inactivation (Fig. 1). The dissociation constant was 5 + 2 mM and the maximum inactivation rate 0.117 + 0.010 min-] at 30°C. At a concentration of 4-methylene-Lproline higher than 24 mM, the inactivation rate decreased. Proline dehydrogenase activity in the incubation medium at 30°C was stable up to 20 min and then decreased. No activity was recovered when the modified mitochondria was washed in order to eliminate the inactivator. The same treatment with the native mitochondria led to a 20% activity loss. The catalytic event appeared to be necessary to inactivate proline dehydrogenase. Indeed, any compound leading to a diminution of the oxidation rate of 4-methylene-L-proline induced a decrease of the inactivation rate. The presence of L-Pro, the substrate, in the reaction mixture protected the enzyme completely for few minutes, but later the activity decreased (Fig. 2). The addition of D,L-lactate, a competitive inhibitor ( K i = 0.9 mM) [18] diminished the inactivation rate (Fig. 2). In the same way, the addition of pyrrole-2carboxylic acid, a non-competitive inhitor [17], also led to a decrease of the inactivation rate (results not shown). The same effect was observed by adding potassium cyanide, an inhibitor of the electron transport system (Fig. 3). The protection was not complete, as potassium cyanide does not prevent the electron transfer of the reduced FAD to the first electron acceptor ubiquinone. The addition of glutathione in the incubation medium reduced the inactivation rate (Fig. 3).

18 ¸ 15 12

9' 0 6'

3" 0 o.oo

o.o

o.o

o.o9

1/[4-methylene-L-Pro]

(mM-~)

Fig. 1. Inactivation of proline dehydrogenase of rat liver mitochondria by 4-methylene-L-proline; determination of the maximal inactivation rate (k) and the dissociation constant (Ki). The mitochondria suspension (120/zl) was incubated at 30°C in 0.1 M phosphate buffer (pH 7.4) in the presence of 4-methylene-L-proline (6-20 mM). The final volume was 480/~l. At various times, aliquots (80/.d) were withdrawn to determine the residual activity. The apparent first-order rate constant (kob s) was calculated for each analogue concentration from the slope of the plot log residual activity vs. incubation time [25]. Data are the average of at least two experiments. Error bars represent the standard deviation of the mean.

80 1001 A .,,.,

50

o

ef-

10 Time

t0

(mln)

Fig. 2. Protection of proline dehydrogenase inactivation by 4-methylene-L-proline with L-Pro and D,L-lactate. The influence of L-Pro and D,L-lactate on the inactivation of proline dehydrogenase by 4-methylene-L-proline (20 mM) was studied by determining, as described in Fig. 1, the inactivation rate in the absence ( n ) and in the presence of 10 mM L-Pro ( • ) or 25 mM D,L-lactate ( zx). Data are the average of two experiments. Error bars represent the standard deviation of the mean.

~ Time

" - ~ I Nu-Enz

~COOH ~ "xN/ I H

proline dehydrogenase

8

~'o

1'2

(mln)

the weak oxidation rate of the fluorinated analogues may be due to the electronegativity effect of fluorine, disturbing the catalytic mechanism of the enzyme. The testing of the proline analogue on a mitochondrial suspension did not allow us to determine the effectiveness of this irreversible inhibitor. Indeed, the concentration of proline dehydrogenase is undetermined so that the number of catalytic events per active site could not be obtained. The concentration of oxidised 4-methylene-L-proline (8 in Scheme III) was determined during the inactivation process, using the 2-aminobenzaldehyde method. After complete inactivation of proline dehydrogenase it was about 0.5 mM, in favor of a partition ratio higher than one. 4-methylene L-proline (3) and both isomers of 5ethynyl proline (6 and 7 in Scheme II) were tested in vivo on two fly strains: Drosophila melanogaster strain Canton S and the tsetse fly Glossina pallidipes (results not shown). Neither toxicity nor behavioural anomaly were detected with the three compounds on D. melanogaster. With the tsetse fly, where the acetylenic compounds were ineffective, the administration of 4Nu - Enz

CH2

CH2

~

Fig. 3. Protection of proline dehydrogenase inactivation by 4-methylene-L-proline with potassium cyanide and glutathione. The influence of L-Pro and D,L-lactate on the inactivation of proline dehydrogenase by 4-methylene-L-proline (20 mM) was studied by determining, as described in Fig. 1, the inactivation rate in the absence ([]) and in the presence of 2 mM potassium cyanide (zx) or 1 mM glutathione ( • ) . Data are the average of two experiments. Error bars represent the standard deviation of the mean.

This observation implies that the reaction product is released in part and then later reacts with the enzyme from the solution. All these facts agree with a mechanism-based inhibition of proline dehydrogenase by 4-methylene-L-proline (Scheme liD. The enzyme oxidises the proline analogue to Al-pyrroline-3-methylene-5-carboxylic acid (8 in Scheme III). The electrophilic species reacts with the enzyme partly as soon as it is formed at the active site and partly by coming back from the medium to the active site. To reduce the release of the reactive species in the medium, fluorinated analogues, known to be more reactive [29], were prepared. We have shown that (E)3-deoxy-C3-fluoro-methylene-D-glucose was a mechanism-based inactivator of xylose isomerase with no protection in the presence of cysteine, while the enzyme was partially protected from the inactivation by 3-deoxy-C3-methylene-D-glucose in the presence of the nucleophile [30]. However, in our case the fluoro analogues 4 and 5 were very poor substrates and did not inactivate proline dehydrogenase. The replacement of an hydrogen atom by a fluorine atom should not greatly affect the steric properties of the compounds. Thus,

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~

N

COOH

~ C O O H

+

I H

S

Scheme III.

I

H Inactive enzyme

81 methylene L-proline was lethal. The flies died within 12 h after the ingestion of 250/zg of the proline analogue. The flight capacity did not seem to be impaired. This specificity of the effect of 4-methylene-L-proline indicates, therefore, that further investigation into the inhibition of proline dehydrogenase will prove worthwhile in the pursuit of a means of tsetse fly control. Proline dehydrogenase does not seem to be an essential enzyme in humans. Indeed, type-I hyperprolinemia, associated with a deficiency of proline dehydrogenase, seems to be benign [31]. This would decrease the risk of toxicity of the compound. Concerning this point, another interesting fact is that 4-methylene-Lproline does not seem to be incorporated into protein

[32]. 4-Methylene-L-proline is a natural product, characteristic of rosaceous plants [33]. As other naturally-occurring imino acids, it may act as an anti-metabolite against potential parasites [33]. To our knowledge, the influence of plants producing 4-methylene-L-proline on the development of tsetse flies has not been reported.

Acknowledgements The authors wish to thank Dr. A. Rendo and D. Fillol for their help in the isolation of rat liver mitochondria. The in vivo tests on D. metanogaster were performed by Dr. J.M. Jallon (Laboratoire de Biologie et de G6n6tique Evolutives, Gif-sur-Yvette, France). The proline analogues were tested on Glossina by P.A. Langley (Tsetse Research Laboratory, Department of Veterinary Medecine, Langford House, Langford, Bristol, UK). They are gratefully acknowledged for their help. We thank Dr. J. Wardell for her careful reading of the manuscript.

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3 4 5 6

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