- Email: [email protected]
The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase
Chem.-Biol. Interactions, 34 (1981) 75--83 © Elsevier/North-Holland Scientific Publishers Ltd.
75
THE E F F E C T O F L-2-AMINO-4-METHOXY-trans-3-BUTENOIC ACID ON SERINE H Y D R O X Y M E T H Y L T R A N S F E R A S E M.J. TISDALE Department of Biochemistry, St. Thomas's Hospital Medical School, London, SE1 7EH (United Kingdom)
(Received June 17th, 1980) (Revision received September 29th, 1980) (Accepted October 10th, 1980)
SUMMARY
The turnout growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the Kin-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation o f Walker cells with R o 0 7 - 7 9 5 7 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentrations ~
INTRODUCTION
Serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes the transfer o f the h y d r o x y m e t h y l group from serine to 5,6,7,8-tetrahydrofolic acid and thus provides one-carbon units which are subsequently used for the de novo biosynthesis of purines and thymine [1,2]. The activity of this enzyme is elevated nearly 5-fold in l y m p h o c y t e s from patients with chronic lymphocytic leukaemia relative to normal l y m p h o c y t e s and to a lesser extent in leucocytes of patients with acute myelocytic and acute l y m p h o c y t i c leukaemia [3]. This m a y reflect the increased dependence of t u m o u r cells on one-carbon metabolism. A n u m b e r of studies suggest that neoplastic cells m a y be more susceptible to interference with one-carbon metabolism than normal cells. Whereas normal cells can survive as well in a methionine-depleted medium containing h o m o c y s t e i n e as they can in a methionine-supplemented media, a n u m b e r of animal and human tumours require the presence of pre-
76
CH3OCH=CHcH/NH2 \COOH
CH30\ /H /C : C\ /C020 H
C
I
®~H I
CH2-- B6
II
formed methionine for growth [4,5]. Also one of the m o s t active anti-tumour agents m e t h o t r e x a t e interferes with one-carbon metabolism by blocking the reduction of dihydrofolate and subsequently thymidylate synthetase [6]. Both chronic granulocytic leukaemic and normal marrow cells are unable to synthesize serine from glucose [7] and their serine requirement might be expected to be m e t by the transfer of a single carbon unit to glycine by serine hydroxymethyltransferase. Inhibitors of this enzyme would thus be expected to be anti-tumour agents. L-2-Amino-4~nethoxy-trans-3-butenoic acid (Ro07-7957,I)is an antibiotic produced by Pseudomonas aeruginosa growing on n-paraffins as the sole source of carbon and energy [8]. This agent, which has recently been shown to act as a methionine antagonist is a p o t e n t t u m o u r growth inhibitor in vitro [9]. It also acts as an irreversible inhibitor of aspartate transaminase [10] and t r y p t o p h a n synthase [11] and might also be expected to inhibit other pyridoxal phosphate-dependent enzymes such as serine hydroxymethyltransferase. This substance does n o t contain chemically reactive groups as such, b u t is transformed into a reactive inhibitor by the holoenzyme, which becomes an agent of its own inactivation. In these systems the reactive species is, presumably, a highly reactive Michael acceptor of the t y p e (II) which could react with an active site Lewis base leading to covalent attachm e n t to the enzyme. In the present study the effect of R o 0 7 - 7 9 5 7 on serine hydroxymethyltransferase has been investigated. MATERIALS AND METHODS
Chemicals L-[3-14C] Serine (spec. act., 56 mCi/mmol) was obtained from the Radiochemical Cenlre, Amersham. Pyridoxal 5-phosphate, 5,5
Cell culture Cells were grown in Dulbeco's modified Eagle medium supplemented with 10% foetal calf serum under an atmosphere of 10% CO2 in air and were
77 subcultured twice weekly. Under these conditions the doubling time of Walker carcinoma was 24 h.
Serine hydroxymethyltransferase assay Serine hydroxymethyltransferase was measured by the radioactive assay of Taylor and Weissbach [12]. After washing in 0.9% NaC1, cells were sonicated in 100 mM Tris--HC1 (pH 7.1) and cenirifuged at 3000 × g for 1 h to yield a supernatant fraction which was used in the enzyme assay. Protein was determined by the m e t h o d of L o w r y et al. [13] using bovine serum albumin as a standard. Each assay system contained 0.1 umol L-[3-14C]serine (spec. act., 2 uCi/pmol), 0.1 umol pyridoxal phosphate, 0.8 u m o l (+)-L-tetrahydrofolate, 4.0 p m o l 2-mercaptoethanol, 30 p m o l potassium phosphate (pH 7.4) and supernatant, in a total volume of 0.2 ml. Reactions were initiated by the addition of enzyme and were terminated with 0.3 ml 1.0 M sodium acetate (pH 4.5) followed by 0.2 ml 0.1 M formaldehyde and 0.3 ml 0.4 M dimedon (in 50% ethanol) and the vessels were heated 5 min in a boiling water bath to accelerate formation of the formaldehyde dimedon derivative. The tubes were then cooled 5 min in an ice-bath before the dimedon c o m p o u n d was extracted by vigorous shaking with 5 ml of toluene at room temperature. After centrifugation to separate the phases 3 ml of the upper phase was transferred to scintillation vials and the radioactivity was determined in a toluene/PPO/POPOP scintillation mixture. RESULTS
L-2-Amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) is an effective inhibitor of the growth of the Walker carcinoma in vitro; LDs0 9.5 pg/ml (Fig. 1). L-Serine at concentrations up to 0.5 mM had no effect on growth inhibition b y Ro07-7957. Production of [~4C]H~O from L-[3-~4C]serine by cytosolic extracts of Walker carcinoma increased linearly with reaction time up to 20 min of incubation (Fig. 2) and with enzyme concentration up to 0.3 mg protein. Duplicate determinations agreed within 4%. Enzyme activity in Walker carcinoma was higher than in extracts of normal human embyronic bladder fibroblasts (Fig. 2). When serine concentration was varied at a fixed concentration of tetrahydrofolate (4 mM) classical hyperbolic saturation kinetics resulting in a linear Lineweaver-Burk plot (Fig. 3) were observed and gave a Kin-value for serine of 0.8 mM, a value similar to that found in other mammalian tissues [3,14]. R o 0 7 - 7 9 5 7 acted as a non-competitive inhibitor of Walker carcinoma serine hydroxymethyltransferase with an apparent inhibition constant of 1.3 mM which was similar to the Kin-value for serine. When the enzyme was incubated with Ro07-7957 in the absence of serine inhibition was time-dependent at constant inactivator concentration (Fig. 4). There was no inhibition in the absence of pyridoxal phosphate (apoenzyme) indicating that the inhibitor formed a complex with pyridoxal phosphate. These results suggest that R o 0 7 - 7 9 5 7 acts as a suicide inactivator of serine hydroxymethyltransferase in vitro.
78 0.9
0-8
0-7
0-6
0"5
0-4
0.~
0o;
0-1
!
"//
I
0
1
log [Ro07-7957] j,K j I m l
Fig. 1. Dose-response curve for Ro07-7957 against Walker carcinoma. Cells were grown in duplicate wells (3.5 ml) o f a 24-well plastic plate (Flow Laboratories, Scotland). Cell number was enumerated daily and the fractional inhibition by Ro07-7957 was calculated from the linear part of the growth curve.
70
60
o
F o
50
40
3(]
c
20
•
•
1G
I 0
5
10
15
20
Time (rain)
Fig. 2. Activity o f serine hydroxymethyltransferase from eytosolie extracts of Walker X) L~ld human embryonic fibroblast~ (s *) as a function of incarcinoma (× cubation time.
79
0"2
o
o
O.
~.~
o
x
0
1
2
3
I 4
/S raM-1
Fig. 3. Double reciprocal plot of the interaction of L-serine (0.25--6 mM) with Walker carcinoma serine hydroxymethyltransferase in the absence or in the presence of 0.82 (e e) and 1.65 (o o) mM Ro07-7957.
When Walker cells were incubated for 24 h in the presence of different concentrations of R o 0 7 - 7 9 5 7 the activity of serine hydroxymethyltransferase was increased at all concentrations of drug (Fig. 5), although the increase was greater at lower concentrations ( < 1 0 pg/ml). In the presence of 10 pg]ml of R o 0 7 - 7 9 5 7 enzyme activity increased linearly with incubation time up t o 7 h of incubation (Fig. 6), when the specific activity was approx, twice the starting level. In the presence of a concentration of cycloheximide (10 pg/ml) which caused 94% inhibition o f protein synthesis no increase in serine hydroxymethyltransferase occurred in the presence of Ro07-7957. Cycloheximide alone had no effect on enzyme activity during the course o f the experiment. These results suggest that in vivo inhibition o f serine hydroxymethyltransferase is accompanied by rapid de novo synthesis of new enzyme. DISCUSSION Highly specific irreversible enzyme inhibitors can contain latent reactive groupings which are activated b y the target enzyme. Upon activation, a chemical reaction ensues between the inhibitor and an active site residue or cofactor resulting in the irreversible inhibition of the enzyme. Virtually all the inhibitors of this t y p e are based on the enzymatic production of Michael acceptors by the target enzyme. Such a reaction is possible with pyridoxal
1-0
0.9
0.8
0.7 o c
0-6
0.5 o
04. 0-3
0.2
0.1
10
20
~
~0
~
60
~0
~
90
Time [mini
Fig. 4. Effect o f incubation time on the inactivation o f serine hydroxymethyltransferase by Ro07-7957 (1 raM). Cytosolic extracts o f Walker carcinoma (165 ~g cytosolic protein per time point) were incubated at 37°C with Ro07-7957 in the absence o f serine and the presence (× x ) or absence (o o) o f pyridoxal phosphate (0.5 raM). At the times indicated samples were removed and assayed for e n z y m e activity in the presence o f 0.5 mM L-serine as described in Methods in a total volume o f 200 ul. E n z y m e activity is expressed as a fraction o f controls incubated under the same conditions in the absence o f Ro07-7957. ?
E
% _? c
I'o
~o
~o
2o
Concentration (pg/ml)
Fig. 5. Effect o f 24-h incubation o f exponentially growing Walker cells with various concentrations o f Ro07-7957 on the activity o f serine hydroxymethyltransferase. At the end of the incubation the cells were centrifuged (300 x g for 3 min), washed with 0.9% NaCI and sonicated in 400 ~1 o f the assay buffer. Cytosolic serine hydroxymethyltransferase activity was determined as described in methods.
81
6
¢ /.---o •'~ o
3
o
¢: 2
I
!
I
I
I ~
T i m o (h)
Fig. 6. Time course of the effect o f Ro07-7957 (10 u g / m l ) o n serine hydroxymethyltransferase activity in the presence (o o) or absence (× × ) of cycloheximide (10 ~g/ml). Both drug and cycloheximide were added at time zero and e n z y m e activity was determined as described in the legend to Fig. 5. E n z y m e activity in the presence of cycloheximide alone remained constant at 3.5 ~ 0.5 nmol [14C]H20/min/mg protein throughout the time course o f the experiment.
phosphate-dependent e n x y m e s with L-2-amino-4.methoxy-trans-3-butenoic acid through an intermediate Michael acceptor (II) which probably serves to inactivate serine hydroxymethyltransferase through reaction with a nucleophilic centre at the active site. R a b b i t liver serine hydroxymethyltransferase has been shown [15] to possess t w o sulphydryl groups on each subunit one of which is c a r b o x y m e t h y l a t e d with iodoacetate in the absence of pyridoxal phosphate resulting in a loss of enzymatic activity. This sulphydryl group is probably at the active site. Thus, inhibition by R o 0 7 - 7 9 5 7 m a y involve modification of this active site sulphydryl group. However, the concentration required for in vitro enzyme inhibition is 10-fold greater than the concentration required for an effective growth inhibitory effect. This suggests that growth inhibition by R o 0 7 - 7 9 5 7 is exerted b y a mechanism other than b y interference with serine transhydroxymethylation. This is also implied b y the inability of L-serine to alter the growth inhibitory effect of R o 0 7 - 7 9 5 7 and the lack of effect of this drug on DNA synthesis. Also there is no effect on thymidylate biosynthesis, since at a concentration of 20 ~g/ml R o 0 7 - 7 9 5 7 causes only a 20% inhibition of the incorporation of 6-[3H]deoxyuridine into DNA over a 24-h period. Addition of R o 0 7 - 7 9 5 7 to intact cells, however, causes an increase in serine hydroxymethyltransferase activity which m a y arise either by derepression of enzyme synthesis, activation of latent enzyme, or by binding to serine hydroxymethyltransferase. R o 0 7 - 7 9 5 7 may stabilize the enzyme and retard the rate of turnover. Since there was no increase in enzyme synthesis
82 in t h e p r e s e n c e o f c y c l o h e x i m i d e it suggests t h a t d e p r e s s i o n o f e n z y m e synthesis o c c u r s in t h e p r e s e n c e o f R o 0 7 - 7 9 5 7 , possibly d u e t o i n h i b i t i o n o f existing e n z y m e . A l t h o u g h m o n k e y liver serine h y d r o x y m e t h y l t r a n s f e r a s e has been s h o w n t o be i n h i b i t e d in vitro b y C i b a c r o n Blue 3 G - A [ 1 6 ] n o evidence has been p r e s e n t e d f o r in vivo e n z y m e inhibition. T h e a b o v e results suggest t h a t in v i t r o e n z y m e i n h i b i t i o n m a y n o t be c o n c o m i t a n t with i n h i b i t i o n o f t h e e n z y m e in i n t a c t cells. This m u s t be t a k e n i n t o c o n s i d e r a t i o n in t h e design o f a n t i - c a n c e r c h e m o t h e r a p e u t i c agents a i m e d at interfering with serine metabolism. ACKNOWLEDGEMENTS
This w o r k has been s u p p o r t e d b y a g r a n t f r o m the C a n c e r R e s e a r c h Campaign. REFERENCES
1 M. Friedkin, Enzymatic conversion of deoxyuridylic acid to thymidylic acid and the participation of tetrahydrofolic acid, Fed. Proc., 16 (1957) 183. 2 S.C. Hartman and J.M. Buchanan, Nucleic acids, purines, pyrimidines (nucleotide synthesis), Ann. Rev. Biochem., 28 (1959) 365. 3 J. Thorndike, T-T. Pelliniemi and W.S. Beck, Serine hydroxymethyltransferase activity and serine incorporation in leukocytes, Cancer Res., 39 (1979) 3435. 4 W. Kreis and M. Goodenow, Methionine requirement and replacement by homocysteine in tissue cultures of selected rodent and human malignant and normal cells, Cancer Res., 38 (1978) 2259. 5 R.M. Hoffman and R.W. Erbe, High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1523. 6 M.J. Osborn, M. Freeman and F.M. Huennekins, Inhibition of dihydrofolic acid reductase by aminopterin and amethopterin, Proc. Soc. Exp. Biol. (N.Y.), 97 (1958) 429. 7 J.D. Regan, H. Vodopick, S. Takeda, W.H. Lee and F.M. Faulcon, Serine requirement in leukemic and normal blood cells, Science, 163 (1969) 1452. 8 U. Sahm, G. Knobloch and F. Wagner, Isolation and characterization of the methionine antagonist L-2-andno-4-methoxy-trans-3-butenoic acid from Pseudomonas aeruginosa grown on n-paraffin, J. Antibiot., 26 (1973) 389. 9 M.J. Tisdale, The effect of the methionine antagonist L-2-amino-4-methoxy-trans-3butenoic acid on the growth and metabolism of Walker carcinosarcoma in vitro, Biochem. Pharmacol., 29 (1980) 501. 10 R.R. Rando, N. Relya and L. Cheng, Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxy-trans-3butenoic acid, J. Biol. Chem., 251 (1976) 3306. 11 E.W. Miles, A new type of pyridoxal-P enzyme catalyzed reaction. The conversion of ~,~-unsaturated amino acids to saturated ~-keto acids by tryptophan synthase, Biochem. Biophys. Res. Commun., 66 (1975) 94. 12 R.T. Taylor and H. Weissbach, Radioactive assay for serine transhydroxymethylase, Anal. Biochem., 13 (1965) 80. 13 O.H. Lowry, W.J. Rosenbrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265.
83 14 P.M. Harish Kumar, J.A. North, J.H. Mangum and N.A. Rao, Cooperative interactions of tetrahydrofolate with purified pig kidney serine transhydroxymethylase and loss of this cooperativity in L 1210 turnouts and in tissues of mice bearing these tumours, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1950. 15 Schirch, S. Slagel, D. Barra, F. Martini and F. Bossa, Evidence for a sulphydryl group at the active site of serine transhydroxymethylase, J. Biol. Chem., 255 (1980) 2986. 16 K.S. Ramesh and N.A. Rao, Inhibition of m o n k e y liver serine hydroxymethyltransferese by Cibacron Blue 3G-A, Biochem. J., 187 (1980) 249.
75
THE E F F E C T O F L-2-AMINO-4-METHOXY-trans-3-BUTENOIC ACID ON SERINE H Y D R O X Y M E T H Y L T R A N S F E R A S E M.J. TISDALE Department of Biochemistry, St. Thomas's Hospital Medical School, London, SE1 7EH (United Kingdom)
(Received June 17th, 1980) (Revision received September 29th, 1980) (Accepted October 10th, 1980)
SUMMARY
The turnout growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the Kin-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation o f Walker cells with R o 0 7 - 7 9 5 7 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentrations ~
INTRODUCTION
Serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes the transfer o f the h y d r o x y m e t h y l group from serine to 5,6,7,8-tetrahydrofolic acid and thus provides one-carbon units which are subsequently used for the de novo biosynthesis of purines and thymine [1,2]. The activity of this enzyme is elevated nearly 5-fold in l y m p h o c y t e s from patients with chronic lymphocytic leukaemia relative to normal l y m p h o c y t e s and to a lesser extent in leucocytes of patients with acute myelocytic and acute l y m p h o c y t i c leukaemia [3]. This m a y reflect the increased dependence of t u m o u r cells on one-carbon metabolism. A n u m b e r of studies suggest that neoplastic cells m a y be more susceptible to interference with one-carbon metabolism than normal cells. Whereas normal cells can survive as well in a methionine-depleted medium containing h o m o c y s t e i n e as they can in a methionine-supplemented media, a n u m b e r of animal and human tumours require the presence of pre-
76
CH3OCH=CHcH/NH2 \COOH
CH30\ /H /C : C\ /C020 H
C
I
®~H I
CH2-- B6
II
formed methionine for growth [4,5]. Also one of the m o s t active anti-tumour agents m e t h o t r e x a t e interferes with one-carbon metabolism by blocking the reduction of dihydrofolate and subsequently thymidylate synthetase [6]. Both chronic granulocytic leukaemic and normal marrow cells are unable to synthesize serine from glucose [7] and their serine requirement might be expected to be m e t by the transfer of a single carbon unit to glycine by serine hydroxymethyltransferase. Inhibitors of this enzyme would thus be expected to be anti-tumour agents. L-2-Amino-4~nethoxy-trans-3-butenoic acid (Ro07-7957,I)is an antibiotic produced by Pseudomonas aeruginosa growing on n-paraffins as the sole source of carbon and energy [8]. This agent, which has recently been shown to act as a methionine antagonist is a p o t e n t t u m o u r growth inhibitor in vitro [9]. It also acts as an irreversible inhibitor of aspartate transaminase [10] and t r y p t o p h a n synthase [11] and might also be expected to inhibit other pyridoxal phosphate-dependent enzymes such as serine hydroxymethyltransferase. This substance does n o t contain chemically reactive groups as such, b u t is transformed into a reactive inhibitor by the holoenzyme, which becomes an agent of its own inactivation. In these systems the reactive species is, presumably, a highly reactive Michael acceptor of the t y p e (II) which could react with an active site Lewis base leading to covalent attachm e n t to the enzyme. In the present study the effect of R o 0 7 - 7 9 5 7 on serine hydroxymethyltransferase has been investigated. MATERIALS AND METHODS
Chemicals L-[3-14C] Serine (spec. act., 56 mCi/mmol) was obtained from the Radiochemical Cenlre, Amersham. Pyridoxal 5-phosphate, 5,5
Cell culture Cells were grown in Dulbeco's modified Eagle medium supplemented with 10% foetal calf serum under an atmosphere of 10% CO2 in air and were
77 subcultured twice weekly. Under these conditions the doubling time of Walker carcinoma was 24 h.
Serine hydroxymethyltransferase assay Serine hydroxymethyltransferase was measured by the radioactive assay of Taylor and Weissbach [12]. After washing in 0.9% NaC1, cells were sonicated in 100 mM Tris--HC1 (pH 7.1) and cenirifuged at 3000 × g for 1 h to yield a supernatant fraction which was used in the enzyme assay. Protein was determined by the m e t h o d of L o w r y et al. [13] using bovine serum albumin as a standard. Each assay system contained 0.1 umol L-[3-14C]serine (spec. act., 2 uCi/pmol), 0.1 umol pyridoxal phosphate, 0.8 u m o l (+)-L-tetrahydrofolate, 4.0 p m o l 2-mercaptoethanol, 30 p m o l potassium phosphate (pH 7.4) and supernatant, in a total volume of 0.2 ml. Reactions were initiated by the addition of enzyme and were terminated with 0.3 ml 1.0 M sodium acetate (pH 4.5) followed by 0.2 ml 0.1 M formaldehyde and 0.3 ml 0.4 M dimedon (in 50% ethanol) and the vessels were heated 5 min in a boiling water bath to accelerate formation of the formaldehyde dimedon derivative. The tubes were then cooled 5 min in an ice-bath before the dimedon c o m p o u n d was extracted by vigorous shaking with 5 ml of toluene at room temperature. After centrifugation to separate the phases 3 ml of the upper phase was transferred to scintillation vials and the radioactivity was determined in a toluene/PPO/POPOP scintillation mixture. RESULTS
L-2-Amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) is an effective inhibitor of the growth of the Walker carcinoma in vitro; LDs0 9.5 pg/ml (Fig. 1). L-Serine at concentrations up to 0.5 mM had no effect on growth inhibition b y Ro07-7957. Production of [~4C]H~O from L-[3-~4C]serine by cytosolic extracts of Walker carcinoma increased linearly with reaction time up to 20 min of incubation (Fig. 2) and with enzyme concentration up to 0.3 mg protein. Duplicate determinations agreed within 4%. Enzyme activity in Walker carcinoma was higher than in extracts of normal human embyronic bladder fibroblasts (Fig. 2). When serine concentration was varied at a fixed concentration of tetrahydrofolate (4 mM) classical hyperbolic saturation kinetics resulting in a linear Lineweaver-Burk plot (Fig. 3) were observed and gave a Kin-value for serine of 0.8 mM, a value similar to that found in other mammalian tissues [3,14]. R o 0 7 - 7 9 5 7 acted as a non-competitive inhibitor of Walker carcinoma serine hydroxymethyltransferase with an apparent inhibition constant of 1.3 mM which was similar to the Kin-value for serine. When the enzyme was incubated with Ro07-7957 in the absence of serine inhibition was time-dependent at constant inactivator concentration (Fig. 4). There was no inhibition in the absence of pyridoxal phosphate (apoenzyme) indicating that the inhibitor formed a complex with pyridoxal phosphate. These results suggest that R o 0 7 - 7 9 5 7 acts as a suicide inactivator of serine hydroxymethyltransferase in vitro.
78 0.9
0-8
0-7
0-6
0"5
0-4
0.~
0o;
0-1
!
"//
I
0
1
log [Ro07-7957] j,K j I m l
Fig. 1. Dose-response curve for Ro07-7957 against Walker carcinoma. Cells were grown in duplicate wells (3.5 ml) o f a 24-well plastic plate (Flow Laboratories, Scotland). Cell number was enumerated daily and the fractional inhibition by Ro07-7957 was calculated from the linear part of the growth curve.
70
60
o
F o
50
40
3(]
c
20
•
•
1G
I 0
5
10
15
20
Time (rain)
Fig. 2. Activity o f serine hydroxymethyltransferase from eytosolie extracts of Walker X) L~ld human embryonic fibroblast~ (s *) as a function of incarcinoma (× cubation time.
79
0"2
o
o
O.
~.~
o
x
0
1
2
3
I 4
/S raM-1
Fig. 3. Double reciprocal plot of the interaction of L-serine (0.25--6 mM) with Walker carcinoma serine hydroxymethyltransferase in the absence or in the presence of 0.82 (e e) and 1.65 (o o) mM Ro07-7957.
When Walker cells were incubated for 24 h in the presence of different concentrations of R o 0 7 - 7 9 5 7 the activity of serine hydroxymethyltransferase was increased at all concentrations of drug (Fig. 5), although the increase was greater at lower concentrations ( < 1 0 pg/ml). In the presence of 10 pg]ml of R o 0 7 - 7 9 5 7 enzyme activity increased linearly with incubation time up t o 7 h of incubation (Fig. 6), when the specific activity was approx, twice the starting level. In the presence of a concentration of cycloheximide (10 pg/ml) which caused 94% inhibition o f protein synthesis no increase in serine hydroxymethyltransferase occurred in the presence of Ro07-7957. Cycloheximide alone had no effect on enzyme activity during the course o f the experiment. These results suggest that in vivo inhibition o f serine hydroxymethyltransferase is accompanied by rapid de novo synthesis of new enzyme. DISCUSSION Highly specific irreversible enzyme inhibitors can contain latent reactive groupings which are activated b y the target enzyme. Upon activation, a chemical reaction ensues between the inhibitor and an active site residue or cofactor resulting in the irreversible inhibition of the enzyme. Virtually all the inhibitors of this t y p e are based on the enzymatic production of Michael acceptors by the target enzyme. Such a reaction is possible with pyridoxal
1-0
0.9
0.8
0.7 o c
0-6
0.5 o
04. 0-3
0.2
0.1
10
20
~
~0
~
60
~0
~
90
Time [mini
Fig. 4. Effect o f incubation time on the inactivation o f serine hydroxymethyltransferase by Ro07-7957 (1 raM). Cytosolic extracts o f Walker carcinoma (165 ~g cytosolic protein per time point) were incubated at 37°C with Ro07-7957 in the absence o f serine and the presence (× x ) or absence (o o) o f pyridoxal phosphate (0.5 raM). At the times indicated samples were removed and assayed for e n z y m e activity in the presence o f 0.5 mM L-serine as described in Methods in a total volume o f 200 ul. E n z y m e activity is expressed as a fraction o f controls incubated under the same conditions in the absence o f Ro07-7957. ?
E
% _? c
I'o
~o
~o
2o
Concentration (pg/ml)
Fig. 5. Effect o f 24-h incubation o f exponentially growing Walker cells with various concentrations o f Ro07-7957 on the activity o f serine hydroxymethyltransferase. At the end of the incubation the cells were centrifuged (300 x g for 3 min), washed with 0.9% NaCI and sonicated in 400 ~1 o f the assay buffer. Cytosolic serine hydroxymethyltransferase activity was determined as described in methods.
81
6
¢ /.---o •'~ o
3
o
¢: 2
I
!
I
I
I ~
T i m o (h)
Fig. 6. Time course of the effect o f Ro07-7957 (10 u g / m l ) o n serine hydroxymethyltransferase activity in the presence (o o) or absence (× × ) of cycloheximide (10 ~g/ml). Both drug and cycloheximide were added at time zero and e n z y m e activity was determined as described in the legend to Fig. 5. E n z y m e activity in the presence of cycloheximide alone remained constant at 3.5 ~ 0.5 nmol [14C]H20/min/mg protein throughout the time course o f the experiment.
phosphate-dependent e n x y m e s with L-2-amino-4.methoxy-trans-3-butenoic acid through an intermediate Michael acceptor (II) which probably serves to inactivate serine hydroxymethyltransferase through reaction with a nucleophilic centre at the active site. R a b b i t liver serine hydroxymethyltransferase has been shown [15] to possess t w o sulphydryl groups on each subunit one of which is c a r b o x y m e t h y l a t e d with iodoacetate in the absence of pyridoxal phosphate resulting in a loss of enzymatic activity. This sulphydryl group is probably at the active site. Thus, inhibition by R o 0 7 - 7 9 5 7 m a y involve modification of this active site sulphydryl group. However, the concentration required for in vitro enzyme inhibition is 10-fold greater than the concentration required for an effective growth inhibitory effect. This suggests that growth inhibition by R o 0 7 - 7 9 5 7 is exerted b y a mechanism other than b y interference with serine transhydroxymethylation. This is also implied b y the inability of L-serine to alter the growth inhibitory effect of R o 0 7 - 7 9 5 7 and the lack of effect of this drug on DNA synthesis. Also there is no effect on thymidylate biosynthesis, since at a concentration of 20 ~g/ml R o 0 7 - 7 9 5 7 causes only a 20% inhibition of the incorporation of 6-[3H]deoxyuridine into DNA over a 24-h period. Addition of R o 0 7 - 7 9 5 7 to intact cells, however, causes an increase in serine hydroxymethyltransferase activity which m a y arise either by derepression of enzyme synthesis, activation of latent enzyme, or by binding to serine hydroxymethyltransferase. R o 0 7 - 7 9 5 7 may stabilize the enzyme and retard the rate of turnover. Since there was no increase in enzyme synthesis
82 in t h e p r e s e n c e o f c y c l o h e x i m i d e it suggests t h a t d e p r e s s i o n o f e n z y m e synthesis o c c u r s in t h e p r e s e n c e o f R o 0 7 - 7 9 5 7 , possibly d u e t o i n h i b i t i o n o f existing e n z y m e . A l t h o u g h m o n k e y liver serine h y d r o x y m e t h y l t r a n s f e r a s e has been s h o w n t o be i n h i b i t e d in vitro b y C i b a c r o n Blue 3 G - A [ 1 6 ] n o evidence has been p r e s e n t e d f o r in vivo e n z y m e inhibition. T h e a b o v e results suggest t h a t in v i t r o e n z y m e i n h i b i t i o n m a y n o t be c o n c o m i t a n t with i n h i b i t i o n o f t h e e n z y m e in i n t a c t cells. This m u s t be t a k e n i n t o c o n s i d e r a t i o n in t h e design o f a n t i - c a n c e r c h e m o t h e r a p e u t i c agents a i m e d at interfering with serine metabolism. ACKNOWLEDGEMENTS
This w o r k has been s u p p o r t e d b y a g r a n t f r o m the C a n c e r R e s e a r c h Campaign. REFERENCES
1 M. Friedkin, Enzymatic conversion of deoxyuridylic acid to thymidylic acid and the participation of tetrahydrofolic acid, Fed. Proc., 16 (1957) 183. 2 S.C. Hartman and J.M. Buchanan, Nucleic acids, purines, pyrimidines (nucleotide synthesis), Ann. Rev. Biochem., 28 (1959) 365. 3 J. Thorndike, T-T. Pelliniemi and W.S. Beck, Serine hydroxymethyltransferase activity and serine incorporation in leukocytes, Cancer Res., 39 (1979) 3435. 4 W. Kreis and M. Goodenow, Methionine requirement and replacement by homocysteine in tissue cultures of selected rodent and human malignant and normal cells, Cancer Res., 38 (1978) 2259. 5 R.M. Hoffman and R.W. Erbe, High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1523. 6 M.J. Osborn, M. Freeman and F.M. Huennekins, Inhibition of dihydrofolic acid reductase by aminopterin and amethopterin, Proc. Soc. Exp. Biol. (N.Y.), 97 (1958) 429. 7 J.D. Regan, H. Vodopick, S. Takeda, W.H. Lee and F.M. Faulcon, Serine requirement in leukemic and normal blood cells, Science, 163 (1969) 1452. 8 U. Sahm, G. Knobloch and F. Wagner, Isolation and characterization of the methionine antagonist L-2-andno-4-methoxy-trans-3-butenoic acid from Pseudomonas aeruginosa grown on n-paraffin, J. Antibiot., 26 (1973) 389. 9 M.J. Tisdale, The effect of the methionine antagonist L-2-amino-4-methoxy-trans-3butenoic acid on the growth and metabolism of Walker carcinosarcoma in vitro, Biochem. Pharmacol., 29 (1980) 501. 10 R.R. Rando, N. Relya and L. Cheng, Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxy-trans-3butenoic acid, J. Biol. Chem., 251 (1976) 3306. 11 E.W. Miles, A new type of pyridoxal-P enzyme catalyzed reaction. The conversion of ~,~-unsaturated amino acids to saturated ~-keto acids by tryptophan synthase, Biochem. Biophys. Res. Commun., 66 (1975) 94. 12 R.T. Taylor and H. Weissbach, Radioactive assay for serine transhydroxymethylase, Anal. Biochem., 13 (1965) 80. 13 O.H. Lowry, W.J. Rosenbrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265.
83 14 P.M. Harish Kumar, J.A. North, J.H. Mangum and N.A. Rao, Cooperative interactions of tetrahydrofolate with purified pig kidney serine transhydroxymethylase and loss of this cooperativity in L 1210 turnouts and in tissues of mice bearing these tumours, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1950. 15 Schirch, S. Slagel, D. Barra, F. Martini and F. Bossa, Evidence for a sulphydryl group at the active site of serine transhydroxymethylase, J. Biol. Chem., 255 (1980) 2986. 16 K.S. Ramesh and N.A. Rao, Inhibition of m o n k e y liver serine hydroxymethyltransferese by Cibacron Blue 3G-A, Biochem. J., 187 (1980) 249.