AHCHIVES OF BIWHEMISTHY Vol.
188, No. 1, May,
Synthesis
AND BIOPHYSICS pp. 137-144, 1978
and Properties
A Transition-State
Analog
of 6-W(Phosphonacetyl)-L-Ornithine Inhibitor
NICHOLAS Department
of Biochemistry,
La Trohe
of Ornithine
Transcarbamylase
J. HOOGENRAAD Uniuersity,
Bundoora,
Received October 13, 1977; revised January
Victoria,
3083, Australia
11, 1978
Procedures are described for the synthesis of S-N-(phosphonacetyl)-L-ornithine from o(CBZ-L-ornithine and from copper-ornithine and phosphonacetic acid. &PALO is a stable analog of the transition state in the reaction catalyzed by ornithine transcarbamylase from rat liver. S-PALO is a competitive inhibitor of ornithine transcarbamylase with respect to carbamyl-P, but not to L-ornithine, indicating that the inhibitor binds to the same form of the enzyme as carbamyl-I’, whereas &PALO and I.-ornithine combine with different forms of the enzyme. Replots of l/u versus concentration of S-PALO are linear with either carbamyl-P or L-ornithine as variable substrate. The inhibition constant (K,) for S-PALO with ornithine transcarbamylase is 2.7 x lo-’ M at 30°C and pH 7.4 in 50 mM Tris-HCl. Thus S-PALO binds some 250 times more tightly than its competing substrate, carbamyl-P. Although S-PALO is a powerful inhibitor of ornithine transcarbamylase, it did not significantly inhibit, citrulline biosynthesis in isolat,ed rat liver mkochondria or urea biosynthesis in intact rat hepatocytes. This failure to inhibit was shown to be due to the permeability barrier imposed by the mitochondrial membranes, since citrulline biosynthesis by mitochondria modified with digitonin or Triton X-100 was substantially inhibited by low concentrations of S-PALO. -
It is widely accepted that enzymes catalyze reactions via activated complexes of the substrates involved in the reaction. Pauling (1) recognized that, since the enzyme binds the activated complexes more tightly than it binds the substrates, analogs of these activated complexes should be potent enzyme inhibitors. A number of these transition-state analogs have thus far been synthesized, and an explicit rationale for their action as enzyme inhibitors has been developed [for recent reviews, see (2, 3)]. It is predicted not only that transition-state analogs should bind tightly to enzymes, but also that inhibition should be highly specific since the activated complexes of substrates are unlikely to be identical for different enzyme reactions. Transition-state analogs have been useful in the elucidation of enzyme reaction mechanisms. For example, Collins and Stark (4) synthesized N-(phosphonacetyl)L-aspartate for studies on aspartate transcarbamylase from Escherichia coli and
Hoogenraad (5) used the same compound for studies on the reaction mechanism of aspartate transcarbamylase from mouse spleen. These compounds are also potentially useful as antimetabolites, and Stark and co-workers (6-9) have shown that the transition-state analog inhibitor of aspartate transcarbamylase effectively blocks de nouo pyrimidine nucleotide biosynthesis and cellular proliferation. Transition-state analogs are also potentially valuable in metabolic modeling studies. The effect of inhibition of one metabolic pathway on other metabolic processes, such as occurs in many inborn errors of metabolism, can be investigated in isolated cells, tissues, or experimental animals using potent, highly specific inhibitors. With this aim in mind, a transition-state analog inhibitor of ornithine transcarbamylase (EC 2.1.3.3) was synthesized based on the methods of Collins and Stark (4) for the synthesis of N-(phosphonacetyl)-L-aspartate. The compound, S-N- (phosphonacetyl) -L-orni-
137 0003-9861/78/1881-0137$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
138
NICHOLAS
J. HOOGENRAAD
thine, combines most of the structural features of the two substrates or the two products of the ornithine transcarbamylase reaction into a single molecule (Fig. 1). Mori et al. (10) have recently published an independent report on the inhibition of beef liver ornithine transcarbamylase by SPALO.’ However, a method for the synthesis of S-PALO or its characterization has not been reported, nor have any data been published on the effects of &PALO on urea biosynthesis in uiuo. In this report, two synthetic schemes, one of which is suitable for the synthesis of radioactively labeled S-PALO, and some of the features of SPALO’s interaction with ornithine transcarbamylase are described. Data are also presented on the effect of S-PALO on urea biosynthesis in isolated rat hepatocytes and on citrulline biosynthesis in isolated rat liver mitochondria. MATERIALS
AND
METHODS
Materials [‘C]Carbamyl-P (16.5 mCi/mmol) and I.-[G-“HIornithine (2.05 Ci/mmol) were purchased from New England Nuclear Corp. [‘4C]Carbamyl-P was purified as described by Adair and Jones (11) to remove an unacceptable quantity of radioactive impurity. The [‘4C]carbamyl-P was stored in small aliquots in a liquid NS freezer. I.-Ornithine hydrochloride, carbamyl-P (dilithium salt), Trisma base [N-tris(hydroxymethyl)methylglycine], and I,-citrulline were obtained from Sigma Chemical Co. a-CBZ-I>-Ornithine was from Fox Chemical Co. and triethyl ester of phosphonacetic acid was from Aldrich Chemical Co. Thionyl chloride was from British Drug Houses. Bio-Rad AGl-X4 (200-400 mesh), chloride form, was from Bio-Rad Laboratories, chelating resin (50-100 mesh) from Sigma Chemical Co., and Sephadex G-10 from Pharmacia. PEI-cellulose plates were purchased from Brinkman Industries. Triton X-100 was purchased from Sigma and digitonin from Hycel Inc., Houston, Texas. Methods
of isolated hepatocytes, mitochonPreparation dria, and mitoplasts. Female Wistar rats of 200-250 g, fasted overnight, were used for the preparation of hepatocytes and liver mitochondria. Hepatocytes were prepared by the method of Berry and Friend (12). The viability of cells was 85597% as judged by staining with 0.02% trypan blue. The rate of glucose formation from ’ Abbreviations used: S-PALO, S-N-(phosphonacetyl)-r.-ornithine; PEI, polyethyleneimine.
FIG. 1. The structures of IS-PALO and the substrates and products of the reaction catalyzed by ornithine transcarbamylase. 10 mM lactate was 1.8-3.2 nmol/min/mg of protein at 37°C. Mitochondria and mitoplasts consisting of digitonin extracts of mitochondria were prepared as described by Clarke (13). Ornithine transcarbamylase assay. Ornithine transcarbamylase was purified to homogeneity from rat liver by the method of Clarke (14). Enzyme was diluted for use in enzyme assays in 10 mM EDTA containing 0.05% (w/v) bovine serum albumin and 1 mM P-mercaptoethanol. Enzyme was assayed at 30°C for 5 min in 50 mM Tris-HCl (pH 7.4) by measuring the conversion of [‘4C]carbamyl-P to [‘4C]citrulline as described by Goldstein et al. (15). In the standard assay, used in the synthesis and isolation of &PALO, [14C]carbamyl-P was 0.12 mM and ornithine was 5 mM. In all assays, less than 5% of the limiting substrate was utilized. Ornithine determination. The L-ornithine content of &PALO was determined enzymatically using an excess of [“C]carbamyl-P after acid hydrolysis in 6 M HCI. Samples of &PALO (approx 1 pmol on a totalphosphorus basis) were hydrolyzed in sealed, evacuated glass tubes at IOO’C for 16 h. The acid hydrolysates were dried in cacao over NaOH pellets and dissolved in 1 ml of 1 M Tris-HCl, pH 8.0. The conversion of [Y!]carbamyl-P to [r4C]citrulline in the presence of the acid hydrolysate (IO ~1) was compared with the conversion in the presence of known amounts of r.-ornithine (between 5 and 50 nmol) and a fixed amount of [‘Clcarbamyl-P (0.5 pmol). The ornithine content of &PALO was corrected for destruction of ornithine on hydrolysis as estimated by controls containing both &PALO and a known amount of added L-ornithine. Analytical procedures. Phosphonacetic acid was prepared from the triethyl ester by hydrolysis in refluxing 6 M HCl for 3 h and recrystallization from glacial acetic acid. Phosphorus was determined quantitatively by the method of Ames and Dubin (16) and phosphorus-containing compounds were detected on PEI-cellulose plates by the method of Bandurski and Axelrod (17). Radioactive samples were counted in the
ANALOG
INHIBITOR
OF ORNITHINE
scintillation cocktail of Anderson and McClure (18) in a Packard Tri-Carb scintillation spectrometer, Model 527. Tritium was counted with an efficiency of 15.8%, and carbon-14 with an efficiency of 85.5%. Copper-r$H]ornithine was synthesized by the method of Ledger and Stewart (19). To 10 mmol of I.-ornithine hydrochloride was added 50 PCi of I.-[G‘Hlornithine. The ornithine was dissolved in boiling water, and basic copper carbonate (3 g) was added slowly. After cooling, the copper-ornithine was isolated by ethanol fractionation and crystallization from aqueous solution. Microanalysis was carried out by the Australian Microanalytical Service, Melbourne. Synthesis of S-PALO (i) Synthesis from a-CBZ-ornithine. Eight-tenths gram of o-CBZ-ornithine (3 mmol) was dissolved in 1 liter of a 50% (v/v) aqueous solution of 1,4-dioxan by stirring at room temperature overnight. Phosphonacetyl chloride (6 mmol) was prepared as described by Collins and Stark (4) and added rapidly, dropwise from an addition funnel, to the solution of a-CBZ-ornithine, kept at pH 9-10 with 2 M NaOH. The sample was dried and the residue was resuspended in 100 ml of water and redried. The sparing solubility of the CBZ derivatives of &PALO and ornithine in water was utilized to remove salt and phosphonacetic acid. The residue was washed with cold water until no more chloride ions or phosphorus was removed in the filtrate. The residue was dried in uacuo over PzO5, after which it was dissolved in a minimum volume of 30% HBr in glacial acetic acid (approx 5 ml). The sample was stirred for 16 h at 4°C to completely hydrolyze the u-CBZ group, after which it was added dropwise to 100 ml of dry (sodium wire) ether at 4°C in glass, screw-capped centrifuge tubes. The pellet of ornithine and S-PALO sedimented on centrifugation at 2000 g and was washed twice with dry ether to remove traces of the brown bromo derivative of CBZ. Care was taken during the washing procedure to keep moisture from the precipitate, since the white solid readily takes up moisture to become an oil. The precipitate was dissolved in 150 ml of 50 mM Tris-HCI, pH 8.0, and run onto a column (30 cm x 8 cm’) of AGI-X4 (chloride form) preequilibrated with 50 mM Tris-HCI, pH 8.0. The column was washed with a further 150 ml of buffer and then eluted with a gradient of O-O.9 M NaCl (500 ml of each). Fractions of 6.7 ml were collected and assayed for total phosphorus content. The capacity of 10 ~1 of a I:100 dilution of each fraction to inhibit the activity of ornithine transcarbamylase was also measured using the standard assay procedure. Fractions containing &PALO were combined, concentrated to 8 ml by rotary evaporation, adjusted to pH 2 with 2 M HCl, and desalted on a column (45 cm X 4 cm’) of Sephadex G-IO. Fractions of 5.4 ml were collected and spot-tested for S-PALO using ninhydrin and the phosphorus detection system of Bandurski
139
TRANSCARBAMYLASE
and Axelrod (17) and for NaCl with AgNO:j (3 g/liter). The desalted sample was neutralized with 1 M LiOH, concentrated by rotary evaporation, and dried in U~CUOover P,O,. The final product was an oil with a faint brown color. (iti Synthesis from copper-(G- ‘Hjornithine. Six millimoles of phosphonacetyl chloride, prepared as described above and dissolved in 20 ml of dry dioxan, was added dropwise to 3 mmol of copper-[“Hlornithine (5.24 x IO” cpm) in 25 ml of water. The pH was maintained at 9-10 with 2 M NaOH. The reaction mixture was dried and redissolved in 10 ml of 0.5 M HCI. It was then passsed through a column (30 cm x 6 cm’) of chelating resin and eluted with water. The eluate was desalted on a column of Sephadex G-10 as described above and applied to a column of AGI-X4 (chloride form) in 150 ml of 50 mM Tris-HCI, pH 8. The sample was chromatographed using a NaCl gradient as described above and fractions containing 6. [“HIPAL (1.63 X 10” cpm) were desalted as described previously (1.51 X IO” cpm). RESULTS
AND
DISCUSSION
Synthesis and Purification of S-PALO Ion-exchange chromatography on AGlX4 (chloride) following synthesis from cyCBZ-ornithine shows a single peak of phosphorus at fraction 50 (Fig. 2), which corresponds exactly to the peak of inhibition of ornithine transcarbamylase. Phosphonacetic acid, which normally elutes with a peak in fraction 62, is not present in this sample since it was removed by washing the sparingly soluble (w-CBZ-&PALO with water. L-Ornithine does not bind to AG-1 under the conditions used, and passes through when the sample is applied to the column. Thus, a simplified procedure for the isolation of S-PALO synthesized by this route is to separate unreacted ornithine from S-PALO using a batchwise procedure, whereby the S-PALO is eluted from AG-1 using 1 M formic acid, followed by removal of formic acid in uacuo over NaOH pellets. Ion-exchange chromatography on AGl following synthesis of &[“H]PALO from copper-[“Hlornithine results in two peaks of phosphorus at fractions 50 and 62 (Fig. 3). The peak at fraction 62 eluted in an identical position to pure phosphonacetic acid. The peak of phosphorus at fraction 50 coincided exactly to the peak of tritium (Fig. 3). Likewise, the ornithine transcarbamylase inhibition profile (not shown)
140
NICHOLAS
J. HOOGENRAAD
Fm. 2. Elution profne of &PALO synthesized from a-CBZ ornithine. A sample was applied to a column (30 X 8 cm) of AG l-X4 resin, 200-400 mesh (chloride form), equilibrated with 50 mM Tris-HCl, pH 8, and eluted with a linear gradient of 500 ml each of the same buffer and 0.9 M NaCl in 50 mM buffer (-----). The fraction size was 6.7 ml. Small aliquots were assayed for phosphorus (M) and ornithine transcarbamylase inhibition (A-A) as described in the text.
5t ‘:- 40 ” 3. E 0 2-
0
IO
20
30
40
50
FRACTION
60
70
so
90
100
NUMBER
FIG. 3. Elution profile of &[“H]PALO synthesized from copper-[“Hlornithine. Conditions identical to those in Fig. 2. Small samples of each fraction were analyzed for tritium (O----0) phosphorus (A-A).
also superimposes on the tritium profile. Free [3H]ornithine (3.64 x lo6 cpm) passed through the column unretarded. When 6r3H]PAL0 was mixed with &PALO synthesized from cu-CBZ-ornithine, the two samples cochromatographed on AGl. Chromatography on Sephadex-GlO resulted in a peak of radioactivity coinciding with the ninhydrin-positive fractions which eluted ahead of the AgN03-precipitable material. The overall yield of &PALO was 16% based on cY-CBZ-ornithine and 23% based on Cu-[3H]ornithine. &PALO has been synthesized from a-CBZ-ornithine on four occasions and from copper-ornithine on fourteen occasions. The concentration of S-PALO which inhibited ornithine transcarbamylase by 50% under the conditions of the standard assay ranged from 1 to 6.5 X lop7 M for these preparations. Microanalysis of 6-[3H]PAL0 (trilithium
were and
salt), C7H,20sNzPLi3(272). Calculated: C, 30.9; H, 4.45; 0,35.3; N, 10.3; P, 11.4. Found: C, 28.7; H, 4.91; 0, 38.8; N, 9.48; P, 10.0. Thus the ratio of C:H:O:N:P was found to be 6.51:13.4:6.60:1.84:0.88. Since H and 0 are slightly higher than expected, it appears that the sample contained a small amount of water. On acid hydrolysis, there was an equimolar ratio of phosphorus and L-ornithine (1.13:1). Chromatography of a 20 mM solution of 6-[3H]PAL0 on PET-cellulose, using 0.4 M LiCl as solvent, resulted in a single spot of organic phosphorus with an Rf of 0.97. This compared with an Rf of 0.71 for (10 mM) trilithium phosphonacetate and an Rf of 0.56 for (5 mM) NaH2P04. There was a single spot of tritium on PEI-cellulose, moving with an R/ of 0.93. In order to verify that the product in the synthesis is S-PALO and not (Y-PALO, the
ANALOG
INHIBITOR
OF ORNITHINE
ninhydrin color yield of PALO was compared with that of L-ornithine. The molar color yield of ornithine was found to be 2.04 x lo4 A.ml/meq, compared with a color yield of 1.96 x lo4 A.ml/meq of phosphorus for PALO. Since the &amino group of ornithine makes a negligible contribution to the color yield (20), the similar color yield for ornithine and PALO confirms that the product is the S isomer. On two occasions, synthesis from copper-[3H]ornithine resulted in the formation of more than one compound containing tritium and phosphorus. There were two main peaks on ion-exchange chromatography, one peak corresponding to &PALO (fraction 50) and having a potent capacity to inhibit ornithine transcarbamylase, and a second peak corresponding to phosphonacetate (fraction 62). This second peak did not inhibit ornithine transcarbamylase and had an apparent molecular weight of 900-1000 on Bio-Gel-P2 when compared with standards consisting of glucose polymers. On PEI-cellulose with 0.4 M LiCl, the compound had an Rf of 0.11, and on acid hydrolysis, it contained an equimolar ratio of ornithine and phosphorus. Based on this preliminary investigation, it appears as if this second product consists of 3 units of &PALO. Stability of &PALO in the Presence Ornithine Transcarbamylase
of
Ornithine transcarbamylase, 2.24 mg/ml, in 0.05% bovine serum albumin, 10 mM EDTA, and 1 mM 2-mercaptoethanol in 50 mM Tris-HCl, pH 7.4, was incubated at 30°C for 5 days in the presence of 2 x 10e4 M S-[“HIPALO. Greater than 98% of the tritium was removed by passage of the sample through Bio-Rad AGl-X4 (chloride) at pH 8.0. Consequently, &PALO was not hydrolyzed to ornithine and phosphonacetate in the presence of ornithine transcarbamylase. Effect of Carbamyl-P PALO
on Binding
of S-
The data in Fig. 4 shows that &PALO is a competitive inhibitor of ornithine transcarbamylase with respect to carbamyl-P, and this confirms the finding of Mori et al.
141
TRANSCARBAMYLASE
(10). The fixed concentration of L-ornithine was essentially saturating, being 10 mM. Consequently, the K,,, obtained in the absence of &PALO is close to the true K, for carbamyl-P at pH 7.4. The value of 8 x lo-” M is close to that obtained by Marshall and Cohen (21) for beef liver ornithine transcarbamylase at pH 7.88 in the presence of 5 mM ornithine. The double-reciprocal plots also show a slight concave downward trend, which was found by Marshall and Cohen (21) to be due to multiple binding sites for carbamyl-P, with different dissociation constants for different binding sites. Replots of l/v versus the concentration of S-PALO were linear, and the relationship of -(x intercept) K’ = (1 + [carbamyl-PI/K,) gave K, values which ranged between 2.2 and 3.4 x 10m7 M. Thus &PALO has an affinity for ornithine transcarbamylase approximately 250 times greater than that of carbamyl-P. Effect of L-Ornithine PALO
on Binding
of 6-
S-PALO is a noncompetitive inhibitor of ornithine transcarbamylase with respect to L-ornithine (Fig. 5). Replots of l/u versus the concentration of inhibitor were linear.
---71
r-F-’
-2
0
2
4
6
9
I/CARBAMYL-P
IO
12
14
I6
(mM)-’
FIG. 4. Effect of carbamyl-P on inhibition of ornithine transcarbamylase by &PALO. Enzyme activity was assayed with I.-ornithine at 10 mM, as described under Methods. The concentration of enzyme used was 10 ng of protein per assay. The concentrations of &PALO were: (O-----3), zero; (a-a), 5 X lo-’ M; (-),
lo-”
M; (.---.),
2 X 10 -’ M.
142
NICHOLAS
J. HOOGENRAAD
FIG. 6. Ordered mechanism for rat liver ornithine transcarbamylase. A, First substrate, carbamyl-P; B, second substrate, ornithine; I, &PALO.
FIG. 5. Effect of r,-ornithine on inhibition of ornithine transcarbamylase by S-PALO. Enzyme activity was measured with carbamyl-P at 2.7 x 10m4M, as described under Methods. The concentrations of 6PALO were: (M), zero; (A-A), 1 x 10-l’ M; (a---17), 2.5 X lo--” M; (.-----.), 5 X 10-l’ M.
The noncompetitive pattern indicates that S-PALO and L-ornithine combine with different forms of the enzyme, whereas the competitive pattern obtained with carbamyl-P suggests that S-PALO and carbamylP compete for the same form of the enzyme. The result is consistent with an ordered mechanism proposed by Marshall and Cohen (21), in which carbamyl-P binds first (Fig. 6). The noncompetitive pattern obtained with ornithine as variable substrate requires that the inhibitor and substrate bind simultaneously to the active site of the enzyme, forming the complex EIB (Fig. 6). Thus, the data fit the rate equation in the forward direction V -=1+i+= u
Ka
Kt,
where K, = K,,,/Kl and Ki, = K-l/K], showing a competitive inhibition pattern with carbamyl-P and a noncompetitive pattern with ornithine. However, it should be emphasized that, based on the limited kinetic analysis presented in this paper, it is not possible to conclude that the reaction mechanism of ornithine transcarbamylase is in fact ordered, since the data presented fit equally well to a random mechanism. Certainly, the rate equation shown above does not explain all of the experimental
observations, since the nonlinear reciprocal plots obtained at high concentrations of carbamyl-P (Fig. 4) have been suggested to be due to multiple binding sites for carbamyl-P (21). Ornithine was found to be an inhibitor of ornithine transcarbamylase even at low concentrations of ornithine (not shown). This substrate inhibition becomes pronounced as the pH is increased and is more severe at low concentrations of the second substrate, carbamyl-P, than at saturating concentrations of carbamyl-P [also shown by Snodgrass (22)]. The data shown in Fig. 5 were obtained over a range of L-ornithine concentrations where no inhibition was encountered. In fact, if anything, double-reciprocal plots are slightly concave downward, a result similar to that obtained with subsaturating concentrations of the second substrate, carbamyl-P, by Marshall and Cohen (21). Snodgrass (22) has proposed that the substrate inhibition is due to the isoelectric form of L-ornithine, which is also the substrate for the reaction. As the pH of the incubation medium increases, the concentration of the isoelectric form of L-ornithine increases, thereby inhibiting the enzyme. Marshall and Cohen (21) have concluded that the reactive species must therefore be that with the S-amino group unionized, since only analogs with a charged a-amino group are effective inhibitors of ornithine transcarbamylase (e.g., norvaline, L-leucine, or a-L-aminobutyric acid). The results presented with S-PALO confirm this conclusion, since the S-amino group of ornithine has been sequestered as an acyl derivative in the transition-state analog. Effect of S-PALO on Urea Biosynthesis in Isolated Rat Hepatocytes In order to test the effectiveness of SPALO as an inhibitor of ornithine transcarbamylase in intact cells, the urea biosyn-
ANALOG
INHIBITOR
OF ORNITHINE
thesis was measured in isolated hepatocytes from fasted rats. The rate of urea formed in the absence of &PALO is shown in Table I, using NH&l and glutamine as substrates. The addition of &PALO at 1 and 10 mM levels had no appreciable effect on the rate of urea synthesis. The amount of S-PALO taken up by the hepatocytes was measured using S-[“HIPALO (1.75 x 10” cpm/mmol). Incubations in the presence of 10 mM &PALO (8.75 x lo4 cpm) resulted in an uptake of 1 pmol (1.7 x lo” cpm). Since S-PALO must enter the mitochondrial matrix in order to inhibit ornithine transcarbamylase in intact cells, experiments were performed with isolated mitochondria to determine if &PALO could pass through the mitochondrial membranes. When mitochondria were incubated with 10 mM S-[“HIPALO, there was no measurable uptake of the inhibitor. Since the limit of detection is around 0.05 to 0.1 pmol of &PALO, more detailed studies on the uptake of S-PALO await the synthesis of S-PALO of high specific activity. Effect of S-PALO on Citrulline Biosynthesis in Isolated Rat Liver Mitochondria Mitochondria were incubated as described for isolated hepatocytes. In order to test whether outer and inner mitochondria TABLE EFFECT
I
OF S-PALO ON UHEA FORMATION ISOI.ATE~) RAT HEPATWYTES”
IN
Rate of urea formation (nmol/min/mg of protein) Substrate
NH,Cl (10 mM) NH,Cl (10 mM) plus Na-lactate (5 mM) Glutamine (10 mM)
No PALO
PALO
PALO
8.06 8.93
8.10 10.94
8.63 8.63
11.82
12.08
10.07
ImM
10 rnM
” Hepatocytes were incubated for 1 h at 37°C in a shaking water bath in Krebs-Ringer bicarbonate solution. The cells (6.6 x IOh-2.1 x lo7 viable cells) were incubated in a total volume of 5 ml in the presence of the substrates shown. After 1 h at 37”C, samples were rapidly cooled to 4°C and cells were removed by centrifugation. Cell-free samples were deproteinized using HClO, and urea was measured using the method of Archibald (23).
143
TRANSCARBAMYLASE
membranes impose a significant barrier to entry of &PALO into the matrix, mitochondria were modified with digitonin and Triton X-100. The extent of modification of the mitochondria can be judged from the effect of added N-acetylglutamate on citrulline biosynthesis (Table II). In intact added N-acetylglutamate mitochondria, stimulated citrulline synthesis fourfold. In mitoplasts the stimulation was 11-fold, and in Triton X-loo-treated mitochondria it was 16-fold. Likewise, the inhibition of citrulline synthesis by B-PALO reflects the degree of modification of the mitochondrial membrane: There is no inhibition in intact mitochondria, 80% inhibition in digitoninTABLE
II
E:FFF:C.I. OF S-PALO ON CITHIW.INE FORMATION ISOI,ATEI~ RAT LIWX MITOCHONDHIA”
Additions
Rate of citrulline tion (pmol/min/mg tein) No PALO
Normal mitochondria None N-Acetylglutamate (1 mM) Digitonin-treated mitochondria None N-Acetylglutamate (1 mM) Triton X-loo-treated mitochondria None N-Acetylglutamate (1 mM)
1 mM
IN
formaof proPALO
4.0 17.0
6.3 15.6
9.3 100.7
8.6 20.9
9.3 148.1
1.0 16.6
” Mitoplasts were prepared by treating a 100 mg/ml suspension of mitochondria with digitonin at 0.15 mg/mg of protein and stirred at 4°C for 15 min. The sample was then diluted threefold with buffer and centrifuged at 10,OOOg for 10 min. The pellet was washed once before suspension in buffer to the original sample volume. Mitochondria were also treated with 0.5% Triton X-100 (v/v) in order to solubilize matrix enzymes. Citrulline synthesis by mitochondrial preparations (4.3-12.5 mg/3-ml incubation) was carried out in the presence of Krebs-Ringer bicarbonate solution containing 10 mM NH&l, 5 mM ATP, 7.5 mM MgClz, 5 mM I.-ornithine, and N-acetylglutamate and S-PALO as indicated. Citrulline formed in a 15-min incubation was measured on HClO, deproteinized samples by the method of Ceriotti and Spandrio (24) as modified by Prescott and Jones (25).
144
NICHOLAS
J HOOGENRAAD
treated mitochondria, and 89% inhibition in Triton X-loo-treated mitochondria. Thus it appears that, although &PALO can pass through the plasma membrane, the mitochondrial membranes impose a significant barrier to the entry of &PALO and prevent it from inhibiting urea biosynthesis in intact cells. In contrast to these results, Mori et al. (10) claim that preliminary experiments showed that the administration of &PALO to mice decreased their tolerance to ammonia. It is possible that &PALO may affect some mechanism other than citrulline biosynthesis in liver, since a report by Howell et al. (26) suggests that the addition of PALO to mammalian cells in culture not only inhibits growth, but leads to cell death. However, since neither report describes the synthesis or characterization of &PALO, it is difficult to explain the basis of these results. Based on the results presented here, 6PALO is a potent inhibitor of ornithine transcarbamylase, and consequently is potentially useful in kinetic and physical studies of ornithine transcarbamylase. Despite these inhibitory properties, its use as a compound useful for studies on the regulation of carbamyl-P synthesis in animals with separate intra- and extramitochondrial carbamyl-P pools will depend on being able to modify &PALO to enable it to enter into the mitochondria matrix. Studies are in progress on substitution of &PALO to a form capable of passage through the mitochondrial membrane. ACKNOWLEDGMENTS I acknowledge the valuable advice to Dr. George Stark, Stanford University, in the synthesis of S-PALO and Drs. Robert Scopes and Geoffrey Howlett, La Trobe University, in the critical reading of this manuscript. This work was carried out with the expert technical assistance of Mrs. T. Sutherland, and was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES 1. PAULING,
L.
(1946)
Chem.
Eng.
News
24,
1375-1377. 2. LINDQIJIST, G. E. (1975) in Drug Design (Ariens, E. J., ed.), Vol. 5, pp. 24-80, Academic Press, New York. 3. WOLFENDEN, R. (1976) Annu. Reu. Biophys. Bioeng. 5, 271-306. 4. COXINS, K. D., AND STAHK, G. R. (1971) J. Biol. Chem. 246, 6599-6605. 5. HOOGENRAAD, N. J. (1974) Arch. Biochem. Biophys. 161, 176-182. 6. Swvnur>, E. A., SF,AVEH, S. S., AND STAIZK, G. R. (1974) J. Biol. Chem. 249,6945-6950. 7. YOSHIDA, T., STARK, G. R., AND HOOGENHAAD, N. J. (1974) J. Biol. Chem. 249,6951-6955. 8. KEMPE, T. D., SWYRYD, E. A., B~JIST, M., AND STARK, G. R. (1976) Cell 9, 541-550. 9. JOHNSON, R. K., INOUYE, T., GOI,IIIN, A., AND STARK, G. R. (1976) Cancer Res. 36,2720-2725. 10. MORI, M., AOYAGI, K., TATIRANA, M., ISHIKAWA, T., AND ISHII, H. (1977) Biochem. Biophys. Res. Commun. 76,900-904. 11. ADAm, L. B., AND JONES, M. E. (1972) J. Biol. Chem. 247,2308-2315. 12. BERRY, M. N., AND FRIEND, D. S. (1969) J. Cell Biol. 43, 506-520. 13. CLARKE, S. (1976) J. Biol. Chem. 251, 1354-1363. 14. CLARKE, S. (1976) Biochem. Biophys. Res. Commun. 71, 1118-1124. 15. GOLDSTEIN A. J., HOOGENHAAD, N. J., JOHNSON, J. D., KIJKANAGA, K., SWIEHCZEWSKI, E., CANN, H. M., AND SUNSHINE, P. (1974) Pediat. Res. 8, 5-12. 16. AMES, B. N., AND DUBIN, D. T. (1960) J. Biol. Chem. 235, 769-775. 17. BANDURSKI, R. S., ANII AXIXHOII, B. (1951) J. Biol. Chem. 193,405-410. 18. ANDERSON, L. E., AND MCCNJHE, W. 0. (1973) Anal. Biochem. 51, 173-179. 19. Lmxxx~, R., AND STEWART, F. M. C. (1965) AZ&. J. Chem. 18,933-935. 20. FRIEDMAN, M., ANII WII.I.IAMS, L. D. (1974) Bioorg. Chem. 3,267-280. 21. MARSHALL, M., AND COHEN, P. P. (1972) J. Biol. Chem. 247, 1654-1668. 7, 22. SNODGHASS, P. J. (1968) Biochemistry 3047-3051. 23. ARCHIBALD, R. M. (1945) J. Biol. Chem. 157, 507-518. 24. CERIOTTI, G., AND SPANDRIO, L. (1963) Clin. Chim. Acta 8,295-303. 25. PKESCOTT, L. M., AND JONES, M. E. (1969) Anal. Biochem. 32,408-419. 26. HOWELL, R., KNIGHT, D., AND JONES, E. (1976) Fed. Proc. 36, 716 (abstract).
188, No. 1, May,
Synthesis
AND BIOPHYSICS pp. 137-144, 1978
and Properties
A Transition-State
Analog
of 6-W(Phosphonacetyl)-L-Ornithine Inhibitor
NICHOLAS Department
of Biochemistry,
La Trohe
of Ornithine
Transcarbamylase
J. HOOGENRAAD Uniuersity,
Bundoora,
Received October 13, 1977; revised January
Victoria,
3083, Australia
11, 1978
Procedures are described for the synthesis of S-N-(phosphonacetyl)-L-ornithine from o(CBZ-L-ornithine and from copper-ornithine and phosphonacetic acid. &PALO is a stable analog of the transition state in the reaction catalyzed by ornithine transcarbamylase from rat liver. S-PALO is a competitive inhibitor of ornithine transcarbamylase with respect to carbamyl-P, but not to L-ornithine, indicating that the inhibitor binds to the same form of the enzyme as carbamyl-I’, whereas &PALO and I.-ornithine combine with different forms of the enzyme. Replots of l/u versus concentration of S-PALO are linear with either carbamyl-P or L-ornithine as variable substrate. The inhibition constant (K,) for S-PALO with ornithine transcarbamylase is 2.7 x lo-’ M at 30°C and pH 7.4 in 50 mM Tris-HCl. Thus S-PALO binds some 250 times more tightly than its competing substrate, carbamyl-P. Although S-PALO is a powerful inhibitor of ornithine transcarbamylase, it did not significantly inhibit, citrulline biosynthesis in isolat,ed rat liver mkochondria or urea biosynthesis in intact rat hepatocytes. This failure to inhibit was shown to be due to the permeability barrier imposed by the mitochondrial membranes, since citrulline biosynthesis by mitochondria modified with digitonin or Triton X-100 was substantially inhibited by low concentrations of S-PALO. -
It is widely accepted that enzymes catalyze reactions via activated complexes of the substrates involved in the reaction. Pauling (1) recognized that, since the enzyme binds the activated complexes more tightly than it binds the substrates, analogs of these activated complexes should be potent enzyme inhibitors. A number of these transition-state analogs have thus far been synthesized, and an explicit rationale for their action as enzyme inhibitors has been developed [for recent reviews, see (2, 3)]. It is predicted not only that transition-state analogs should bind tightly to enzymes, but also that inhibition should be highly specific since the activated complexes of substrates are unlikely to be identical for different enzyme reactions. Transition-state analogs have been useful in the elucidation of enzyme reaction mechanisms. For example, Collins and Stark (4) synthesized N-(phosphonacetyl)L-aspartate for studies on aspartate transcarbamylase from Escherichia coli and
Hoogenraad (5) used the same compound for studies on the reaction mechanism of aspartate transcarbamylase from mouse spleen. These compounds are also potentially useful as antimetabolites, and Stark and co-workers (6-9) have shown that the transition-state analog inhibitor of aspartate transcarbamylase effectively blocks de nouo pyrimidine nucleotide biosynthesis and cellular proliferation. Transition-state analogs are also potentially valuable in metabolic modeling studies. The effect of inhibition of one metabolic pathway on other metabolic processes, such as occurs in many inborn errors of metabolism, can be investigated in isolated cells, tissues, or experimental animals using potent, highly specific inhibitors. With this aim in mind, a transition-state analog inhibitor of ornithine transcarbamylase (EC 2.1.3.3) was synthesized based on the methods of Collins and Stark (4) for the synthesis of N-(phosphonacetyl)-L-aspartate. The compound, S-N- (phosphonacetyl) -L-orni-
137 0003-9861/78/1881-0137$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
138
NICHOLAS
J. HOOGENRAAD
thine, combines most of the structural features of the two substrates or the two products of the ornithine transcarbamylase reaction into a single molecule (Fig. 1). Mori et al. (10) have recently published an independent report on the inhibition of beef liver ornithine transcarbamylase by SPALO.’ However, a method for the synthesis of S-PALO or its characterization has not been reported, nor have any data been published on the effects of &PALO on urea biosynthesis in uiuo. In this report, two synthetic schemes, one of which is suitable for the synthesis of radioactively labeled S-PALO, and some of the features of SPALO’s interaction with ornithine transcarbamylase are described. Data are also presented on the effect of S-PALO on urea biosynthesis in isolated rat hepatocytes and on citrulline biosynthesis in isolated rat liver mitochondria. MATERIALS
AND
METHODS
Materials [‘C]Carbamyl-P (16.5 mCi/mmol) and I.-[G-“HIornithine (2.05 Ci/mmol) were purchased from New England Nuclear Corp. [‘4C]Carbamyl-P was purified as described by Adair and Jones (11) to remove an unacceptable quantity of radioactive impurity. The [‘4C]carbamyl-P was stored in small aliquots in a liquid NS freezer. I.-Ornithine hydrochloride, carbamyl-P (dilithium salt), Trisma base [N-tris(hydroxymethyl)methylglycine], and I,-citrulline were obtained from Sigma Chemical Co. a-CBZ-I>-Ornithine was from Fox Chemical Co. and triethyl ester of phosphonacetic acid was from Aldrich Chemical Co. Thionyl chloride was from British Drug Houses. Bio-Rad AGl-X4 (200-400 mesh), chloride form, was from Bio-Rad Laboratories, chelating resin (50-100 mesh) from Sigma Chemical Co., and Sephadex G-10 from Pharmacia. PEI-cellulose plates were purchased from Brinkman Industries. Triton X-100 was purchased from Sigma and digitonin from Hycel Inc., Houston, Texas. Methods
of isolated hepatocytes, mitochonPreparation dria, and mitoplasts. Female Wistar rats of 200-250 g, fasted overnight, were used for the preparation of hepatocytes and liver mitochondria. Hepatocytes were prepared by the method of Berry and Friend (12). The viability of cells was 85597% as judged by staining with 0.02% trypan blue. The rate of glucose formation from ’ Abbreviations used: S-PALO, S-N-(phosphonacetyl)-r.-ornithine; PEI, polyethyleneimine.
FIG. 1. The structures of IS-PALO and the substrates and products of the reaction catalyzed by ornithine transcarbamylase. 10 mM lactate was 1.8-3.2 nmol/min/mg of protein at 37°C. Mitochondria and mitoplasts consisting of digitonin extracts of mitochondria were prepared as described by Clarke (13). Ornithine transcarbamylase assay. Ornithine transcarbamylase was purified to homogeneity from rat liver by the method of Clarke (14). Enzyme was diluted for use in enzyme assays in 10 mM EDTA containing 0.05% (w/v) bovine serum albumin and 1 mM P-mercaptoethanol. Enzyme was assayed at 30°C for 5 min in 50 mM Tris-HCl (pH 7.4) by measuring the conversion of [‘4C]carbamyl-P to [‘4C]citrulline as described by Goldstein et al. (15). In the standard assay, used in the synthesis and isolation of &PALO, [14C]carbamyl-P was 0.12 mM and ornithine was 5 mM. In all assays, less than 5% of the limiting substrate was utilized. Ornithine determination. The L-ornithine content of &PALO was determined enzymatically using an excess of [“C]carbamyl-P after acid hydrolysis in 6 M HCI. Samples of &PALO (approx 1 pmol on a totalphosphorus basis) were hydrolyzed in sealed, evacuated glass tubes at IOO’C for 16 h. The acid hydrolysates were dried in cacao over NaOH pellets and dissolved in 1 ml of 1 M Tris-HCl, pH 8.0. The conversion of [Y!]carbamyl-P to [r4C]citrulline in the presence of the acid hydrolysate (IO ~1) was compared with the conversion in the presence of known amounts of r.-ornithine (between 5 and 50 nmol) and a fixed amount of [‘Clcarbamyl-P (0.5 pmol). The ornithine content of &PALO was corrected for destruction of ornithine on hydrolysis as estimated by controls containing both &PALO and a known amount of added L-ornithine. Analytical procedures. Phosphonacetic acid was prepared from the triethyl ester by hydrolysis in refluxing 6 M HCl for 3 h and recrystallization from glacial acetic acid. Phosphorus was determined quantitatively by the method of Ames and Dubin (16) and phosphorus-containing compounds were detected on PEI-cellulose plates by the method of Bandurski and Axelrod (17). Radioactive samples were counted in the
ANALOG
INHIBITOR
OF ORNITHINE
scintillation cocktail of Anderson and McClure (18) in a Packard Tri-Carb scintillation spectrometer, Model 527. Tritium was counted with an efficiency of 15.8%, and carbon-14 with an efficiency of 85.5%. Copper-r$H]ornithine was synthesized by the method of Ledger and Stewart (19). To 10 mmol of I.-ornithine hydrochloride was added 50 PCi of I.-[G‘Hlornithine. The ornithine was dissolved in boiling water, and basic copper carbonate (3 g) was added slowly. After cooling, the copper-ornithine was isolated by ethanol fractionation and crystallization from aqueous solution. Microanalysis was carried out by the Australian Microanalytical Service, Melbourne. Synthesis of S-PALO (i) Synthesis from a-CBZ-ornithine. Eight-tenths gram of o-CBZ-ornithine (3 mmol) was dissolved in 1 liter of a 50% (v/v) aqueous solution of 1,4-dioxan by stirring at room temperature overnight. Phosphonacetyl chloride (6 mmol) was prepared as described by Collins and Stark (4) and added rapidly, dropwise from an addition funnel, to the solution of a-CBZ-ornithine, kept at pH 9-10 with 2 M NaOH. The sample was dried and the residue was resuspended in 100 ml of water and redried. The sparing solubility of the CBZ derivatives of &PALO and ornithine in water was utilized to remove salt and phosphonacetic acid. The residue was washed with cold water until no more chloride ions or phosphorus was removed in the filtrate. The residue was dried in uacuo over PzO5, after which it was dissolved in a minimum volume of 30% HBr in glacial acetic acid (approx 5 ml). The sample was stirred for 16 h at 4°C to completely hydrolyze the u-CBZ group, after which it was added dropwise to 100 ml of dry (sodium wire) ether at 4°C in glass, screw-capped centrifuge tubes. The pellet of ornithine and S-PALO sedimented on centrifugation at 2000 g and was washed twice with dry ether to remove traces of the brown bromo derivative of CBZ. Care was taken during the washing procedure to keep moisture from the precipitate, since the white solid readily takes up moisture to become an oil. The precipitate was dissolved in 150 ml of 50 mM Tris-HCI, pH 8.0, and run onto a column (30 cm x 8 cm’) of AGI-X4 (chloride form) preequilibrated with 50 mM Tris-HCI, pH 8.0. The column was washed with a further 150 ml of buffer and then eluted with a gradient of O-O.9 M NaCl (500 ml of each). Fractions of 6.7 ml were collected and assayed for total phosphorus content. The capacity of 10 ~1 of a I:100 dilution of each fraction to inhibit the activity of ornithine transcarbamylase was also measured using the standard assay procedure. Fractions containing &PALO were combined, concentrated to 8 ml by rotary evaporation, adjusted to pH 2 with 2 M HCl, and desalted on a column (45 cm X 4 cm’) of Sephadex G-IO. Fractions of 5.4 ml were collected and spot-tested for S-PALO using ninhydrin and the phosphorus detection system of Bandurski
139
TRANSCARBAMYLASE
and Axelrod (17) and for NaCl with AgNO:j (3 g/liter). The desalted sample was neutralized with 1 M LiOH, concentrated by rotary evaporation, and dried in U~CUOover P,O,. The final product was an oil with a faint brown color. (iti Synthesis from copper-(G- ‘Hjornithine. Six millimoles of phosphonacetyl chloride, prepared as described above and dissolved in 20 ml of dry dioxan, was added dropwise to 3 mmol of copper-[“Hlornithine (5.24 x IO” cpm) in 25 ml of water. The pH was maintained at 9-10 with 2 M NaOH. The reaction mixture was dried and redissolved in 10 ml of 0.5 M HCI. It was then passsed through a column (30 cm x 6 cm’) of chelating resin and eluted with water. The eluate was desalted on a column of Sephadex G-10 as described above and applied to a column of AGI-X4 (chloride form) in 150 ml of 50 mM Tris-HCI, pH 8. The sample was chromatographed using a NaCl gradient as described above and fractions containing 6. [“HIPAL (1.63 X 10” cpm) were desalted as described previously (1.51 X IO” cpm). RESULTS
AND
DISCUSSION
Synthesis and Purification of S-PALO Ion-exchange chromatography on AGlX4 (chloride) following synthesis from cyCBZ-ornithine shows a single peak of phosphorus at fraction 50 (Fig. 2), which corresponds exactly to the peak of inhibition of ornithine transcarbamylase. Phosphonacetic acid, which normally elutes with a peak in fraction 62, is not present in this sample since it was removed by washing the sparingly soluble (w-CBZ-&PALO with water. L-Ornithine does not bind to AG-1 under the conditions used, and passes through when the sample is applied to the column. Thus, a simplified procedure for the isolation of S-PALO synthesized by this route is to separate unreacted ornithine from S-PALO using a batchwise procedure, whereby the S-PALO is eluted from AG-1 using 1 M formic acid, followed by removal of formic acid in uacuo over NaOH pellets. Ion-exchange chromatography on AGl following synthesis of &[“H]PALO from copper-[“Hlornithine results in two peaks of phosphorus at fractions 50 and 62 (Fig. 3). The peak at fraction 62 eluted in an identical position to pure phosphonacetic acid. The peak of phosphorus at fraction 50 coincided exactly to the peak of tritium (Fig. 3). Likewise, the ornithine transcarbamylase inhibition profile (not shown)
140
NICHOLAS
J. HOOGENRAAD
Fm. 2. Elution profne of &PALO synthesized from a-CBZ ornithine. A sample was applied to a column (30 X 8 cm) of AG l-X4 resin, 200-400 mesh (chloride form), equilibrated with 50 mM Tris-HCl, pH 8, and eluted with a linear gradient of 500 ml each of the same buffer and 0.9 M NaCl in 50 mM buffer (-----). The fraction size was 6.7 ml. Small aliquots were assayed for phosphorus (M) and ornithine transcarbamylase inhibition (A-A) as described in the text.
5t ‘:- 40 ” 3. E 0 2-
0
IO
20
30
40
50
FRACTION
60
70
so
90
100
NUMBER
FIG. 3. Elution profile of &[“H]PALO synthesized from copper-[“Hlornithine. Conditions identical to those in Fig. 2. Small samples of each fraction were analyzed for tritium (O----0) phosphorus (A-A).
also superimposes on the tritium profile. Free [3H]ornithine (3.64 x lo6 cpm) passed through the column unretarded. When 6r3H]PAL0 was mixed with &PALO synthesized from cu-CBZ-ornithine, the two samples cochromatographed on AGl. Chromatography on Sephadex-GlO resulted in a peak of radioactivity coinciding with the ninhydrin-positive fractions which eluted ahead of the AgN03-precipitable material. The overall yield of &PALO was 16% based on cY-CBZ-ornithine and 23% based on Cu-[3H]ornithine. &PALO has been synthesized from a-CBZ-ornithine on four occasions and from copper-ornithine on fourteen occasions. The concentration of S-PALO which inhibited ornithine transcarbamylase by 50% under the conditions of the standard assay ranged from 1 to 6.5 X lop7 M for these preparations. Microanalysis of 6-[3H]PAL0 (trilithium
were and
salt), C7H,20sNzPLi3(272). Calculated: C, 30.9; H, 4.45; 0,35.3; N, 10.3; P, 11.4. Found: C, 28.7; H, 4.91; 0, 38.8; N, 9.48; P, 10.0. Thus the ratio of C:H:O:N:P was found to be 6.51:13.4:6.60:1.84:0.88. Since H and 0 are slightly higher than expected, it appears that the sample contained a small amount of water. On acid hydrolysis, there was an equimolar ratio of phosphorus and L-ornithine (1.13:1). Chromatography of a 20 mM solution of 6-[3H]PAL0 on PET-cellulose, using 0.4 M LiCl as solvent, resulted in a single spot of organic phosphorus with an Rf of 0.97. This compared with an Rf of 0.71 for (10 mM) trilithium phosphonacetate and an Rf of 0.56 for (5 mM) NaH2P04. There was a single spot of tritium on PEI-cellulose, moving with an R/ of 0.93. In order to verify that the product in the synthesis is S-PALO and not (Y-PALO, the
ANALOG
INHIBITOR
OF ORNITHINE
ninhydrin color yield of PALO was compared with that of L-ornithine. The molar color yield of ornithine was found to be 2.04 x lo4 A.ml/meq, compared with a color yield of 1.96 x lo4 A.ml/meq of phosphorus for PALO. Since the &amino group of ornithine makes a negligible contribution to the color yield (20), the similar color yield for ornithine and PALO confirms that the product is the S isomer. On two occasions, synthesis from copper-[3H]ornithine resulted in the formation of more than one compound containing tritium and phosphorus. There were two main peaks on ion-exchange chromatography, one peak corresponding to &PALO (fraction 50) and having a potent capacity to inhibit ornithine transcarbamylase, and a second peak corresponding to phosphonacetate (fraction 62). This second peak did not inhibit ornithine transcarbamylase and had an apparent molecular weight of 900-1000 on Bio-Gel-P2 when compared with standards consisting of glucose polymers. On PEI-cellulose with 0.4 M LiCl, the compound had an Rf of 0.11, and on acid hydrolysis, it contained an equimolar ratio of ornithine and phosphorus. Based on this preliminary investigation, it appears as if this second product consists of 3 units of &PALO. Stability of &PALO in the Presence Ornithine Transcarbamylase
of
Ornithine transcarbamylase, 2.24 mg/ml, in 0.05% bovine serum albumin, 10 mM EDTA, and 1 mM 2-mercaptoethanol in 50 mM Tris-HCl, pH 7.4, was incubated at 30°C for 5 days in the presence of 2 x 10e4 M S-[“HIPALO. Greater than 98% of the tritium was removed by passage of the sample through Bio-Rad AGl-X4 (chloride) at pH 8.0. Consequently, &PALO was not hydrolyzed to ornithine and phosphonacetate in the presence of ornithine transcarbamylase. Effect of Carbamyl-P PALO
on Binding
of S-
The data in Fig. 4 shows that &PALO is a competitive inhibitor of ornithine transcarbamylase with respect to carbamyl-P, and this confirms the finding of Mori et al.
141
TRANSCARBAMYLASE
(10). The fixed concentration of L-ornithine was essentially saturating, being 10 mM. Consequently, the K,,, obtained in the absence of &PALO is close to the true K, for carbamyl-P at pH 7.4. The value of 8 x lo-” M is close to that obtained by Marshall and Cohen (21) for beef liver ornithine transcarbamylase at pH 7.88 in the presence of 5 mM ornithine. The double-reciprocal plots also show a slight concave downward trend, which was found by Marshall and Cohen (21) to be due to multiple binding sites for carbamyl-P, with different dissociation constants for different binding sites. Replots of l/v versus the concentration of S-PALO were linear, and the relationship of -(x intercept) K’ = (1 + [carbamyl-PI/K,) gave K, values which ranged between 2.2 and 3.4 x 10m7 M. Thus &PALO has an affinity for ornithine transcarbamylase approximately 250 times greater than that of carbamyl-P. Effect of L-Ornithine PALO
on Binding
of 6-
S-PALO is a noncompetitive inhibitor of ornithine transcarbamylase with respect to L-ornithine (Fig. 5). Replots of l/u versus the concentration of inhibitor were linear.
---71
r-F-’
-2
0
2
4
6
9
I/CARBAMYL-P
IO
12
14
I6
(mM)-’
FIG. 4. Effect of carbamyl-P on inhibition of ornithine transcarbamylase by &PALO. Enzyme activity was assayed with I.-ornithine at 10 mM, as described under Methods. The concentration of enzyme used was 10 ng of protein per assay. The concentrations of &PALO were: (O-----3), zero; (a-a), 5 X lo-’ M; (-),
lo-”
M; (.---.),
2 X 10 -’ M.
142
NICHOLAS
J. HOOGENRAAD
FIG. 6. Ordered mechanism for rat liver ornithine transcarbamylase. A, First substrate, carbamyl-P; B, second substrate, ornithine; I, &PALO.
FIG. 5. Effect of r,-ornithine on inhibition of ornithine transcarbamylase by S-PALO. Enzyme activity was measured with carbamyl-P at 2.7 x 10m4M, as described under Methods. The concentrations of 6PALO were: (M), zero; (A-A), 1 x 10-l’ M; (a---17), 2.5 X lo--” M; (.-----.), 5 X 10-l’ M.
The noncompetitive pattern indicates that S-PALO and L-ornithine combine with different forms of the enzyme, whereas the competitive pattern obtained with carbamyl-P suggests that S-PALO and carbamylP compete for the same form of the enzyme. The result is consistent with an ordered mechanism proposed by Marshall and Cohen (21), in which carbamyl-P binds first (Fig. 6). The noncompetitive pattern obtained with ornithine as variable substrate requires that the inhibitor and substrate bind simultaneously to the active site of the enzyme, forming the complex EIB (Fig. 6). Thus, the data fit the rate equation in the forward direction V -=1+i+= u
Ka
Kt,
where K, = K,,,/Kl and Ki, = K-l/K], showing a competitive inhibition pattern with carbamyl-P and a noncompetitive pattern with ornithine. However, it should be emphasized that, based on the limited kinetic analysis presented in this paper, it is not possible to conclude that the reaction mechanism of ornithine transcarbamylase is in fact ordered, since the data presented fit equally well to a random mechanism. Certainly, the rate equation shown above does not explain all of the experimental
observations, since the nonlinear reciprocal plots obtained at high concentrations of carbamyl-P (Fig. 4) have been suggested to be due to multiple binding sites for carbamyl-P (21). Ornithine was found to be an inhibitor of ornithine transcarbamylase even at low concentrations of ornithine (not shown). This substrate inhibition becomes pronounced as the pH is increased and is more severe at low concentrations of the second substrate, carbamyl-P, than at saturating concentrations of carbamyl-P [also shown by Snodgrass (22)]. The data shown in Fig. 5 were obtained over a range of L-ornithine concentrations where no inhibition was encountered. In fact, if anything, double-reciprocal plots are slightly concave downward, a result similar to that obtained with subsaturating concentrations of the second substrate, carbamyl-P, by Marshall and Cohen (21). Snodgrass (22) has proposed that the substrate inhibition is due to the isoelectric form of L-ornithine, which is also the substrate for the reaction. As the pH of the incubation medium increases, the concentration of the isoelectric form of L-ornithine increases, thereby inhibiting the enzyme. Marshall and Cohen (21) have concluded that the reactive species must therefore be that with the S-amino group unionized, since only analogs with a charged a-amino group are effective inhibitors of ornithine transcarbamylase (e.g., norvaline, L-leucine, or a-L-aminobutyric acid). The results presented with S-PALO confirm this conclusion, since the S-amino group of ornithine has been sequestered as an acyl derivative in the transition-state analog. Effect of S-PALO on Urea Biosynthesis in Isolated Rat Hepatocytes In order to test the effectiveness of SPALO as an inhibitor of ornithine transcarbamylase in intact cells, the urea biosyn-
ANALOG
INHIBITOR
OF ORNITHINE
thesis was measured in isolated hepatocytes from fasted rats. The rate of urea formed in the absence of &PALO is shown in Table I, using NH&l and glutamine as substrates. The addition of &PALO at 1 and 10 mM levels had no appreciable effect on the rate of urea synthesis. The amount of S-PALO taken up by the hepatocytes was measured using S-[“HIPALO (1.75 x 10” cpm/mmol). Incubations in the presence of 10 mM &PALO (8.75 x lo4 cpm) resulted in an uptake of 1 pmol (1.7 x lo” cpm). Since S-PALO must enter the mitochondrial matrix in order to inhibit ornithine transcarbamylase in intact cells, experiments were performed with isolated mitochondria to determine if &PALO could pass through the mitochondrial membranes. When mitochondria were incubated with 10 mM S-[“HIPALO, there was no measurable uptake of the inhibitor. Since the limit of detection is around 0.05 to 0.1 pmol of &PALO, more detailed studies on the uptake of S-PALO await the synthesis of S-PALO of high specific activity. Effect of S-PALO on Citrulline Biosynthesis in Isolated Rat Liver Mitochondria Mitochondria were incubated as described for isolated hepatocytes. In order to test whether outer and inner mitochondria TABLE EFFECT
I
OF S-PALO ON UHEA FORMATION ISOI.ATE~) RAT HEPATWYTES”
IN
Rate of urea formation (nmol/min/mg of protein) Substrate
NH,Cl (10 mM) NH,Cl (10 mM) plus Na-lactate (5 mM) Glutamine (10 mM)
No PALO
PALO
PALO
8.06 8.93
8.10 10.94
8.63 8.63
11.82
12.08
10.07
ImM
10 rnM
” Hepatocytes were incubated for 1 h at 37°C in a shaking water bath in Krebs-Ringer bicarbonate solution. The cells (6.6 x IOh-2.1 x lo7 viable cells) were incubated in a total volume of 5 ml in the presence of the substrates shown. After 1 h at 37”C, samples were rapidly cooled to 4°C and cells were removed by centrifugation. Cell-free samples were deproteinized using HClO, and urea was measured using the method of Archibald (23).
143
TRANSCARBAMYLASE
membranes impose a significant barrier to entry of &PALO into the matrix, mitochondria were modified with digitonin and Triton X-100. The extent of modification of the mitochondria can be judged from the effect of added N-acetylglutamate on citrulline biosynthesis (Table II). In intact added N-acetylglutamate mitochondria, stimulated citrulline synthesis fourfold. In mitoplasts the stimulation was 11-fold, and in Triton X-loo-treated mitochondria it was 16-fold. Likewise, the inhibition of citrulline synthesis by B-PALO reflects the degree of modification of the mitochondrial membrane: There is no inhibition in intact mitochondria, 80% inhibition in digitoninTABLE
II
E:FFF:C.I. OF S-PALO ON CITHIW.INE FORMATION ISOI,ATEI~ RAT LIWX MITOCHONDHIA”
Additions
Rate of citrulline tion (pmol/min/mg tein) No PALO
Normal mitochondria None N-Acetylglutamate (1 mM) Digitonin-treated mitochondria None N-Acetylglutamate (1 mM) Triton X-loo-treated mitochondria None N-Acetylglutamate (1 mM)
1 mM
IN
formaof proPALO
4.0 17.0
6.3 15.6
9.3 100.7
8.6 20.9
9.3 148.1
1.0 16.6
” Mitoplasts were prepared by treating a 100 mg/ml suspension of mitochondria with digitonin at 0.15 mg/mg of protein and stirred at 4°C for 15 min. The sample was then diluted threefold with buffer and centrifuged at 10,OOOg for 10 min. The pellet was washed once before suspension in buffer to the original sample volume. Mitochondria were also treated with 0.5% Triton X-100 (v/v) in order to solubilize matrix enzymes. Citrulline synthesis by mitochondrial preparations (4.3-12.5 mg/3-ml incubation) was carried out in the presence of Krebs-Ringer bicarbonate solution containing 10 mM NH&l, 5 mM ATP, 7.5 mM MgClz, 5 mM I.-ornithine, and N-acetylglutamate and S-PALO as indicated. Citrulline formed in a 15-min incubation was measured on HClO, deproteinized samples by the method of Ceriotti and Spandrio (24) as modified by Prescott and Jones (25).
144
NICHOLAS
J HOOGENRAAD
treated mitochondria, and 89% inhibition in Triton X-loo-treated mitochondria. Thus it appears that, although &PALO can pass through the plasma membrane, the mitochondrial membranes impose a significant barrier to the entry of &PALO and prevent it from inhibiting urea biosynthesis in intact cells. In contrast to these results, Mori et al. (10) claim that preliminary experiments showed that the administration of &PALO to mice decreased their tolerance to ammonia. It is possible that &PALO may affect some mechanism other than citrulline biosynthesis in liver, since a report by Howell et al. (26) suggests that the addition of PALO to mammalian cells in culture not only inhibits growth, but leads to cell death. However, since neither report describes the synthesis or characterization of &PALO, it is difficult to explain the basis of these results. Based on the results presented here, 6PALO is a potent inhibitor of ornithine transcarbamylase, and consequently is potentially useful in kinetic and physical studies of ornithine transcarbamylase. Despite these inhibitory properties, its use as a compound useful for studies on the regulation of carbamyl-P synthesis in animals with separate intra- and extramitochondrial carbamyl-P pools will depend on being able to modify &PALO to enable it to enter into the mitochondria matrix. Studies are in progress on substitution of &PALO to a form capable of passage through the mitochondrial membrane. ACKNOWLEDGMENTS I acknowledge the valuable advice to Dr. George Stark, Stanford University, in the synthesis of S-PALO and Drs. Robert Scopes and Geoffrey Howlett, La Trobe University, in the critical reading of this manuscript. This work was carried out with the expert technical assistance of Mrs. T. Sutherland, and was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES 1. PAULING,
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