The Characteristics and Significance of Sulfonamides as Substrates

THE JOURNAL OF Bro~ocrcn~ CHEMISTRY Vol. 254, No. 20, Issue of October 25, pp. 10337-10345, 1979 Printed in U.S. A.

The Characteristics and Significance of Sulfonamides as Substrates for Escherichia coli Dihydropteroate Synthase*

(Received for publication, May 29, 1979)

Susan Roland,+ Robert Ferone,$ Robert J. Harvey,* Virgil L. Styles&j and Robert W. Morrison$ From the Deuartments of $Microbioloev and SOwanic Chemistry, The Wellcome Research Laboratories, Research Triangle Park, North‘Carolina 27’709

.,_ - Y

Sulfonamides are known to compete with p-aminobenzoic acid for dihydropteroate synthase. Others have reported that some sulfonamides are alternate substrates, but the significance of these observations to the antimicrobial action of sulfonamides has not been studied. We have shown that sulfanilamide, sulfathiazole, and sulfamethoxazole are efficient alternate substrates for this reaction, with apparent K,,, values equivalent to their Ki values as competitive inhibitors.

The products synthesized from the sulfonamides in uitro were chromatographically similar to chemically prepared dihydropterin-sulfonamides. A culture of Escherichiu coli B converted 29% of 0.625 pM [35S]sulfamethoxazole to a product which was identified as dihydropterin-sulfamethoxazole. Greater than 99% of the product was found in the medium and the cellular concentration of radiolabel was 52 pM. This lack of accumulation was consistent with our finding that sulfonamides diffuse into E. coli and that the active transport of [35S]sulfanilamide could not be demonstrated.

The growth rate of E. coli B was not inhibited by 2 pM of chemically synthesized dihydropterin-sulfamethoxazole. No significant inhibition of thymidylate synthase, N’,N”-methylenetetrahydrofolate dehydrogenase, N’,N”-methenyltetrahydrofolate cyclohydrolase, or dihydrofolate reductase was found with the aromatic and dihydropterin-sulfonamides. High concentrations (50 to 150 pM) of some of the compounds were inhibitory to GTP cyclohydrolase, hydroxyme- thyldihydropterin pyrophosphokinase, and serine hydroxymethyltransferase. The dihydropterin-sulfonamides were product inhibitors of dihydropteroate synthase, and were inhibitors of dihydrofolate synthetase. However, to obtain substantial inhibition of these enzymes by the dihydropterin-sulfonamides in duo, higher concentrations of these compounds are required than those which are attainable intracellularly.

These data show that sulfonamides are effective alternate substrates for E. coli dihydropteroate synthase, but that the dihydropterin-sulfonamide products formed do not contribute significantly to the growth inhibition by sulfonamides.

Sulfonamides have been used for many years in clinical practice as effective chemotherapeutic agents. It has been documented that the most sensitive locus of sulfonamide inhibition is competition with p-AB’ for Hzpteroate synthase

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: p-AB, p-aminobenzoic acid; H1- as a

(l-6). In fact, the classic textbook case of competitive inhibition is sulfonamide antagonism of folate biosynthesis (7-11).

The sulfonamides tested against H*pteroate synthase in kinetic studies were shown to be competitive inhibitors with p-AB orp-aminobenzoylglutamic acid when the 7&dihydropterin substrate was in excess. However, Brown (1) and Shiota et al. (2) observed that preincubation of sulfonamides with bacterial extracts and limiting amounts of the 7&dihydropterin resulted in inhibition that could not be overcome by subsequent addition of p-AB. This observation led to the hypothesis that the sulfonamide was depleting the 7,8-dihydropterin pool by reacting as a p-AB substrate analog to form a 7,8-dihydropterin-sulfonamide adduct. Brown (1) observed an additional radioactive spot on chromatograms of reaction mixtures containing [““S]sulfanilic acid, a 7,8-dihydropterin,

Mg’+, and enzyme. He suggested (1) that the spot was the pterin-sulfanilic acid adduct, which may be an effective inhibitor of p-AB utilization.

Additional evidence for the enzymatic synthesis of a pterin- sulfonamide folate analog was obtained by Bock et al. (12). With E. coli extracts and [35S]SMX, they demonstrated an enzymatic product with chromatographic properties identical with authentic H2ptCH2SMX (see Fig. 1). They demonstrated a similar product from E. coli cultures grown with SMX.

Both investigators showed that sulfonamides not only compete with p-AB but are also metabolized in vitro in the presence of Hzpteroate synthase and a 7,8-dihydropterin to a sulfonamide-containing product. Similarly, Swedberg et al. (13) have also recently shown the synthesis of a pterin-sulfathiazole adduct with extracts from E. coli.

In this study we have investigated the significance of the sulfonamide-containing product to the mechanism of action of sulfonamide inhibition. The authentic pterin-sulfonamides were chemically synthesized and were utilized to confirm the identity of the enzymatic product. They were also tested as inhibitors of various folate enzymes and, sometimes, their effects on whole cell growth were determined. We found that sulfonamides are good substrates for H2pteroate synthase and that pterin-sulfonamides are synthesized in uiuo. Several of the enzymes tested were inhibited by the dihydropterin-sulfonamides. However, H2ptCH2SMX synthesized in uiuo diffused out of the cell, and exogenous H2ptCH2SMX added at physiologically significant concentrations did not inhibit cell

prefix before pt, pteroate, or folate, indicates the 7,8-dihvdro forms: Hrfolate, 5,6,7,8-tetrahydrofolate; ptCH*SMX, l-amino% {4-[N-(5: methyl-3-isoxazoyl)sulfamoyl]anilinomethyl}pteridin-4( 3H)-one; ptCHzSA, 2-amino-6-(4-sulfamoylanilinomethyl)pteridin-4(3H)-one; ptCH$TZ, 2-amino-6-[4-(2-thiazoylsulfamoyl)anilinomethyl] pteridin-4(3H)-one; ptCH*OPP, 2-amino-4-oxo-6-hydroxymethylpteridine pyrophosphate; ptCHzOH; 2.amino-4-oxo-6-hydroxymethylpteridine; SMX, sulfamethoxazole; STZ, sulfathiazole; SA. sulfanilamide.

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10338 Sulfonamides as Substrates for Dihydropteroate Synthase

R NAME -

-SO,NH, H,ptCH,SA

H,ptCH,SMX

-SW<;] H,ptCH,STZ

FIG. 1. The structures of the 7,8-dihydropterin-sulfonamides. The pyrazine ring is fully oxidized in the aromatic species.

growth. Thus, we conclude that the observed inhibitions of folate enzymes by the pterin-sulfonamides make no significant contribution to bacterial growth inhibition by sulfonamides.

MATERIALS AND METHODS

Radiochemicals

[carboxy-‘4C]p-AB (IO and 55 Ci/mol) was purchased from ICN Pharmaceuticals, Cleveland, 0. L-[U-‘%]Glutamic acid (260 Ci/mol) was purchased from Schwartz/Mann, Orangeburg, N. Y. [3H]H20 (1 mCi/g), [‘%]dextran (1.125 mCi/g), and L-[U-‘%]leucine (308 Ci/ mol) were purchased from New England Nuclear, Boston, Mass. L-[3- “‘C]Serine (54 Ci/mol), [3-?l]sulfamethazole (sulfamethoxazole) (9.1 to 28 Ci/mol), [35S]sulfanilamide (18.6 Ci/mol), [2-%]sulfathiazole (2.1 Ci/mol), and 2-amino-&oxo-4,5-diamino[2-‘“Clpyrimidine were purchased from Amersham, Arlington Heights, Ill.

Biochemicals

Folate was purchased from Calbiochem, LaJolla, Calif. Pteroate was a sift from Dr. Charles M. Baueh. Universitv of South Alabama. Mobile, Ala. All other biochemical: were from Sigma Chemical Co.; St. Louis, MO. unless otherwise specified. All chemicals were of reagent grade.

Preparation of Pteridines

2-Amino-6-(4-sulfamoylanilinomethyl)pteridin-4(3H)-one (ptCH#A) (see Fig. I)-Under a nitrogen atmosphere, a mixture of 0.55 g (1.6 mmol) of 2,4-diamino-6-(4-sulfamoylanilinomethyl)- pteridine (14) and 140 ml of 0.2 N NaOH was heated at reflux for 2 h to give a greenish yellow solution which was then adjusted to pH 5 with glacial acetic acid. The yellow precipitate was collected by suction fntration, washed sequentially with water and methanol, and dried under vacuum (70°C): yield 0.49 g (88%); m.p. >3OO”C, NMR (MegSC+‘Hs) S 4.49 (d, J = 6 Hz, 2H), 6.68 (d, J = 9 Hz, 2H), 6.90 (br s, 4H), 7.09 (t, J = 6 Hz, IH), 7.50 (d, J = 9 Hz, 2H), 8.63 (s, IH), 11.51 (br s, 1H); UV A,., (0.1 N NaOH) 257 nm (E 36,200), 364 (E 8,500); mass spectrum, m/e 347 (M).

Calculated C 44.95, H 3.77, N 28.23, S 9.23 Found: C 44.67, H 3.80, N 28.14, S 9.14

This method of synthesis represents an improvement over previously reported procedures (15-17) because of its unequivocal nature and high yield.

2-Amino-6-[4-(2-thiazoylsulfamoyl)anilinomethyl]pteridin-4(3H)- one (ptCHkZ’Z)2 (see Fig. 1)-A mixture of 0.70 g (3.0 mmol) of 2-

’ Attempts to couple either STZ or SMX by the method of Taylor (14) resulted in the formation of the wrong isomer, evidently because of enhanced acidity of the sulfonamide functionality. The described method is a modification of the procedure of Roberts and Shealy (18).

acetylamino-6-formyl-pteridin4(3H)-one (19), 1.0 g (3.9 mmol) of STZ, 20 ml of anhydrous dimethyl sulfoxide, and 2.0 g of 3 A molecular sieves stood at room temperature under nitrogen for 20 h. The solution was pipetted from the sieves and concentrated under high vacuum (0.1 torr; 45°C bath). To the glassy residue, dissolved in 100 ml of anhydrous dimethylformamide, was added 0.14 g of sodium borohydride. The orange solution was stirred under nitrogen at room temperature for 22 h before the solvent was again removed under high vacuum. While still under nitrogen, the residue was dissolved in 750 ml of deoxygenated 0.1 N NaOH and the solution was stirred at 80°C for 15 min before being adjusted to pH 5 with glacial acetic acid. The precipitate was collected by Ntration under nitrogen, washed sequentially with water and methanol, and dried under vacuum (70°C): yield 0.46 g of the crude pteridine. A 54-mg sample of this material was subjected to DEAE-cellulose chromatography to separate the ptCH&STZ from highly fluorescent contamination (2). The fractions containing the purified ptCHzSTZ (based on TLC and UV) were pooled and adjusted to pH 5 with glacial acetic acid. The yellow precipitate was isolated by centrifugation, washed with water, and dried under vacuum (70°C): yield 21 mg; m.p. >3OO”C; NMR (MeSO--‘H6) 6 4.46 (d, J = 6 Hz, 2H), 6.65 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 4.5 Hz, lH), 6.85 (br s, 2H), 7.08 (br t, J = 6 Hz, IH), 7.16 (d, J = 4.5 Hz, IH), 7.50 (d, J = 8.7 Hz, 2H), 8.63 (s, lH), 11.3 (br s, lH), 12.1 (br s, IH); UV h,,, (0.1 N NaOH) 257 nm (E 37,606), 271 sh (32,700), 360 (9,600); mass spectrum, m/e 430 (M).

Calculated: C 42.85, H 3.60, N 24.99 Found: C 42.73, H 3.63, N 24.91

2.Amino-6- (-4-[N- (5.methyl-3-isoxazoyl)sulfamoyl]anilinomethyl)pteridin4(3H)-one @tCH$MX)‘-By the preceding method, with 2-acetylamino-6-formylpteridin4(3H)-one (0.58 g, 2.5 mmol) and SMX (0.70 g, 2.8 mmol), 0.26 g of crude solid was produced from which 0.13 g of the pure ptCHSMX was obtained after two recrys- tallizations from methanol. NMR (MeSO-‘He) 6 2.26 (s, 3H), 4.47 (d, J = 6 Hz, 2H), 6.05 (s, IH), 6.69 (d, J = 8.8 Hz, 2H), 6.87 (br s, 2H), 7.25 (t, J= 6 Hz, lH), 7.52 (d, J= 8.8 Hz, 2H), 8.64 (s, lH), 11.15 (br, 2H); UV h,., (0.1 N NaOH) 261 nm (e 32,400), 365 (8,700); mass spectrum, m/e 428 (M).

Calculated: C 45.73, H 4.06, N 25.10 Found: C 45.56, H 3.67, N 25.28

An alternative synthesis of this compound has been published (12). The 7,8-dihydro derivatives of pteroate, folate, ptCH*OPP, [2-

‘%]ptCHzOH, and the pterin-sulfonamides were prepared by sodium dithionite reduction (20). Evidences for successful reduction of the pterin-sulfonamides were a UV spectral shift characteristic of reduced pterins and folates (21), the visual change under UV light (254 nm) from UV-absorbing material (aromatic pterins) to UV blue fluorescing material (7,8-dihydropterins), and the fact that 7,8-dihydropterin- sulfonamides were substrates for rat liver H*folate reductase, whereas the aromatic species were not.” We assumed an extinction coefficient of 30,ooO at 255 to 265 nm in 0.1 N NaOH to determine the concentration of the 7,8-dihydropterin-sulfonamides.

H,folate was purchased from Sigma Chemical Co., St. Louis, MO. N”,N”-Methylene H.,folate was prepared as described by Friedkin (22). N’,N”-Methenyl H,folate was prepared from N”-formyl Hlfolate by acidic crystallization following the method of Rabinowitz (23). [2- ‘C]ptCH,OH (36 Ci/mol) was prepared by a condensation of dihy- droxyacetone with 2-amino-6-oxo-4,5-diamino[2-“‘C]pyrimidine (24) and ptCHzOPP prepared as in Ref. 25.

Enzyme Assays

1. GTP cyclohydrolase (EC 3.5.4.16) was assayed by the method of Brown (26). The enzyme, isolated from E. coli B, was purified 2000- fold by GTP-agarose affinity chromatography.”

2. H2ptCH20H pyrophosphokinase (EC 2.7.6.3) was assayed with [2-‘%]H2ptCH20H (10 Ci/mol) in reaction mixtures similar to those prepared by Richey and Brown (27). The enzyme, isolated from E. coli B was purified IOOO-fold. Aliquots of each reaction mixture were spotted on MN PEI-cellulose plates (Brinkman) and developed as- cendingly in 0.05 M Tris-HCl, pH 8.6 (25’C), containing 1 M NaCl. The area containing the product, HzptCHzOPP, was cut out and

’ J. Nixon, personal communication.

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Sulfonamides as Substrates for Dihydropteroate Synthase 10339

analyzed for radioactivity as described previously (5). The apparent K, value for HzptCHzOH was 0.7 PM.~

3. Hzpteroate synthase (EC 2.5.1.15) was assayed with [carboxy- “‘C]p-AB (IO Ci/mol) (25). The enzyme was a partially purified E. coli B preparation eluted from a Sephadex G-100 column (27) with a specific activity of 1 to 2 nmol/min/mg of protein.

4. Hzfolate synthetase (EC 6.3.2.12) was assayed with L-[U-~“C]- glutamic acid (1.25 Ci/mol) according to the procedure of Webb and Ferone (28). The enzyme source was the same as for Hgpteroate synthase.

5. Hpfolate reductase (EC 1.5.1.3) was assayed spectrophotometri- tally according to the procedure of Baccanari et al. (29) with the use of enzyme from E. coli RT500 purified to homogeneity by methotrex- ate affinity chromatography. Rat liver dihydrofolate reductase was purified by Sephadex G-100 column (30) chromatography.

6. Serine hydroxymethyltransferase (EC 2.1.2.1) was assayed with L-[3-‘4C]serine (0.2 Ci/mol) according to the method of Taylor and Weissbach (31). E. coli B cells (stored at -7O”C), purchased from the Grain Processing Corp., Muscatine, Ia., were suspended in 0.05 M Tris-HCl buffer, pH 8.3 (25’C), and ruptured by passage twice through a French pressure cell (Aminco) at 11,000 p.s.i. The 200,000 x g (1 h) supernatant fluid was used as the enzyme source.

7. Thymidylate synthetase (EC 2.1.1.45) was assayed spectropho- tometrically by the method of Friedkin (22) with a 200,000 x g (1 h) supernatant fluid of a crude preparation of E. coli B cells harvested from glucose minimal medium (32) as the source of enzyme.

8. N”,N’O-methylene-Hlfolate dehydrogenase (EC 1.5.1.5) was assayed by the method of Scrimgeour and Huennekens (33) except that N”,N”‘-methylene Hlfolate was prepared prior to assay, and 0.03 M

Tris/maleate, pH 8.5;buffer was used. The enzyme source was a partially purified E. coli B enzyme eluted from a DEAE-cellulose column (34).

9. N”,N’“-methenyl-H,folate cyclohydrolase (EC 3.5.4.9) was assayed according to the method of Greenberg (35). The enzyme source was the same as for serine hydroxymethyltransferase.

E. coli Growth Cultures for Permeability Studies

E. coli ML30 cultures (Am = 1.3 to 1.5) were harvested and the cells were washed with 50 mM phosphate, pH 7.0, buffer before being used for diffusion studies. Cultures of cells (100 ml) prepared for active transport studies (E. coli ML30 or E. coli B S5206) were harvested when the Am reached 0.4. These cells were washed and resuspended in 50 mM phosphate, pH 7.0, buffer to an Aem of 8 to 14.

E. coli Growth Inhibition Studies

Aerobic cultures containing 40 ml of glucose minimal medium and 0.025 to 4 mM SA, or 0.5 to 100 pM SMX, or 0.25 to 10 pM STZ, or 5 to 34 PM ptCH&MX, or no drug were inoculated with an overnight culture of E. coli S5206 (initial A m = 0.01-0.03). The cultures were incubated at 37°C and the growth rate was monitored to a final Aa, 5 0.6. There was a l%- to 2-h delay before the inhibited growth rate was established in the cultures with sulfonamides.

To maintain the stability of H2ptCH2SMX, growth inhibition studies were performed anaerobically with a Tris medium containing per liter: 12.1 g of Tris base, 0.136 g of KHzPO+ 0.68 mg of CaC12, 2.5 mg of MgC12. 6H20, 0.02 mg of FeC12. 4H20, and 0.4 g of (NH,)&O, with the pH adjusted to pH 7.4 with HCl.

Thunberg cuvettes contained 2.5 ml of double strength medium, H20, E. coli B S5206 (Am = 0.035), 0.6% glucose, 1.0% sodium ascorbate, 1 mM dithiothreitol, and the contents of the side arm (0.5 ml) in a total of 5 ml. The side arm of the control cuvette contained 0.6% sodium ascorbate and the side arm of the test cuvette contained 0.6% sodium ascorbate and 20 PM HzptCHzSMX. The cuvettes were incubated 30 min at 33’C prior to tipping in the contents of the side arm. The incubation was continued and the growth rate was monitored until the Arjoo 5 0.6.

In Vivo Synthesis ofptCHzSMX

A IOO-ml culture containing Tris medium, 0.5% glucose, 0.7 pM [3- ““S]SMX (5.4 Ci/mol), and E. coli B S5206 (A, = 0.094) was incubated 17 h at 30°C under a nitrogen sparge. One liter of the same medium was then inoculated with this E. coli B culture (initial Am was 0.033) and incubated at 30°C for 19 h under a nitrogen sparge. The cells (Aw = 0.420) were collected by centrifugation (Sorvall GSA rotor) at 23,300 x g for 50 min at ambient temperature. The culture

’ R. Ferone and P. Novak, unpublished data.

supernatant (785 ml) was adjusted to pH 7.0 and pumped at 40 ml/h onto a column (2.5 x 30 cm) of DE52 (Whatman, Clifton, N. J.) at 5”C, equilibrated with 0.01 M Tris-Cl, pH 8.0 at 5°C. A linear gradient (800 ml of buffer, 0 to 1.0 M KCI) was applied to the column, followed by 1500 ml of buffer with 1.0 M KCl. Fractions (12 to 15 ml) were collected and samples of each were analyzed for radioactivity and were chromatographed to evaluate the contents of the peaks. The fractions of interest were pooled and applied at 60 ml/h to a column (0.7 x 3.0 cm) containing 5 mg of acid-washed Norit A (activated charcoal) layered over 10 mg of CFll cellulose (Whatman). The column was washed with 50 ml of H20. The bound material was eluted with 6 ml of N&0H:H20:EtOH (1:4:5). The eluate was concentrated to dryness by rotary evaporation at 4O’C. The residue was dissolved in 3 to 4 ml of H20 and the solution was concentrated again. The residue was dissolved in a small volume of 0.1 N NaOH and stored at -70°C.

In Vivo Concentration ofptCH2SMX

A 20-ml culture of E. coli B S5206 (Am = 0.3), grown aerobically in Tris medium, was added to 100 ml of Tris medium containing 0.625 PM [3-“5S]SMX (28 Ci/mol), and was incubated at 30°C with shaking. The extracellular concentration of products was measured when 2-ml samples of the growth culture were removed at 30-min intervals and passed through a 25.mm HA Millipore filter that had previously been washed with 3 ml of Tris medium. Before the entire sample had ffitered, the filtrate was removed and samples (100 ~1) were analyzed chromatographically. The cultures were also used to determine the intracellular concentration of products. At 30-min intervals, medium (3 ml, 30°C) was applied to a prewashed fdter. A 2-ml sample of the growth culture was added and the total 5 ml was filtered almost to dryness. An additional 3 ml of medium (30°C) was added to wash the cells and just before the filters became dry, the vacuum was removed. The filters were removed and analyzed for radioactivity in vials containing 10 ml of Bray’s (New England Nuclear) scintillation fluid. The calculation of the cellular concentrations assumes that 1 ml of a culture at an AMxl of 1.0 contains 0.6 ~1 of intracellular water (36).

Chromatography

Paper Chromatography-Chromatograms (3 x 20 cm, Whatman No. 3MM) were developed descendingly with 0.1 M Sorenson’s PO,, pH 7.0 buffer, to a length of 18 cm from the origin. The dried chromatograms were cut into l-cm sections which were analyzed for radioactivity in vials containing 10 ml of a mix of 15.2 g 2,5-bis-2(5-t- butylbenzoxazoylyl)-thiopene/gal toluene.

Thin Layer Chromatography-Thin layer chromatography was performed on cellulose (20 x 20 cm) (0.25 mm) plates (Machery and Nagel, Germany). Chromatograms were developed for 12 cm in Na2HP04 (5%, w/v), LiCl (0.21%, w/v), or NKCl (3%, w/v). Chro- matograms of reaction mixtures were scanned with a thin layer scanner (model LB 2760, Berthold, West Germany).

High Performance Liquid Chromatography Techniques

Reverse phase high performance liquid chromatography was performed on a PBondapak C,S column (0.78 x 30 cm) in a model ALC/ GPC 244 unit equipped with model 6000A pumps and a model 660 solvent programer (all from Waters Associates, Milford, Mass.). Peaks were detected by a Waters Associates model 440 UV detector (X = 254 nm) and a model FS-970 continuous flow fluoresence monitor (Schoeffel Instrument Co., Westwood, N. J.) with an excitation wavelength set at 350 nm and an emission filter (KV 418) with a cutoff below 410 nm. The fluorescence monitor was used in a 1 @A range with a sensitivity setting of approximately 51 and a time constant at 6 s. The column was developed with a linear gradient of 0 to 60% methanol in water at an approximate flow rate of 1 ml/min. Fractions (0.5 ml) were collected and aliquots (50 ~1) were analyzed for radioactivity in 10 ml of Bray’s scintillation fluid. The volume of samples injected ranged from 2.5 to 55 ~1.

Transport Studies

Active transport functions were assayed according to the method of Kaback (37) except that whole cells were used and 10 mM glucose was provided as an energy source. Reaction mixtures (100 ~1) were incubated at 31°C in 0.05 M 4-(2-hydroxyethyl)-l-piperazinethanesul- fonic acid buffer, pH 7.2, over a 3-minute time course.

The diffusion of solutes into cells was determined from a study of solute uptake as described by Black and Gerhardt (38). The solute “space” was determined by the relationship:

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where v, is the known volume of the solution added to packed cells, 0.5 ml in this study; v, is the known volume of packed cells; Co is the solute concentration of v,; Cfis the final concentration of solute in the presence of cells; and S”’ is per cent of cell pack penetrated. This method is based on the dilution of radiolabel of the solute by the intracellular water of a packed cell pellet compared to the dilution of [3H]H,0 by these cells. When S” is determined with [“H]H20 as solute, it is a measure of the extracellular and the intracellular space. When S” is determined with [‘4C]dextran (Mr = 20,ooO) as solute, it represents the total extracellular space. The difference between these values is the intracellular water space.

Miscellaneous Methods Radioactivity was determined by the use of a Beckman LS-230

liquid scintillation spectrometer. Ultraviolet spectra were recorded by either a Cary 118 or Gilford 240 spectrophotometer. NMR spectra were determined on a Varian XL-100 spectrometer by the Fourier transform method. Field desorption mass spectral data were obtained with a Varian MAT 731 spectrometer. Elemental microanalyses were performed by Atlantic Microlab, Inc., Atlanta, Ga. Specific growth rates and EDso values were calculated as described previously (39). Cbromatograms of standards (5 to 10 nmol) were viewed under UV light (254 nm) to locate the spots.

Enzyme kinetic data were evaluated with computer programs for the analysis of competitive and noncompetitive inhibitors (40). Eval- uations were made at two inhibitor concentrations compared to the control. Apparent K,, values were calculated from the slope (K,/ V,,,,,) of plots of I/[ V] versus l/[S]. Apparent K,, values were calculated from the intercept of the l/[ V] axis of double reciprocal plots. Kinetic studies were conducted with assay procedures adequate to insure the measurement of initial velocities (25).

Protein was determined by the method of Lowry et al. (41), with bovine serum albumin as standard.

RESULTS

Enzymatic Product Formation from Radioactive Sulfon- amides-The enzymatic synthesis of a product containing a radioactive sulfur moiety was demonstrated when Hnpteroate synthase was incubated with [3-“‘S]SMX (9 Ci/mol) as substrate in place of ID-AB. When an aliquot (100 ~1) of each reaction mixture was chromatographed on paper, a peak of radioactivity at an RF value of 0.23 was evident (Fig. 2A). This peak represents 64% of the total radioactivity and it is not present in a control reaction mixture lacking H2ptCH20PP. The radioactive material migrated to the same position as authentic H2ptCH2SMX (Fig. 20). The small peak at an RF value of 0.45 represents ptCH*SMX. No precautions were taken to limit oxidation during chromatography, although the reaction mixtures were incubated in the presence of 5 mM dithiothreitol.

To demonstrate that the product contained a pteridine moiety, radioactive dihydropteridine substrate, [2-“‘Cl- H2ptCH20PP, was enzymatically synthesized in situ from [2- ?]H2ptCH20H (10 Ci/mol); Hnpteroate synthase and SMX were then added, and the reaction mixtures were incubated an additional period. The chromatogram of the complete reaction indicates a peak at RF value of 0.24 (54% of the total radioactivity) that is identical with the RF value for the radioactive peak demonstrated from the reaction mixtures incubated with [3-“5S]SMX and for authentic H2ptCH2SMX. The chromatogram of the control reaction mixture, lacking Hspteroate synthase, shows two peaks and a shoulder (Fig. 2B). The peak at RF value of 0.58 corresponded to [2-r4C]- ptCHzOH and remained unchanged throughout the enzymatic incubation and chromatography. The peak at an RF value of 0.73 co-chromatographs with HzptCHzOPP and represents the product of HzptCHzOH pyrophosphokinase (the substrate for Hzpteroate synthase). Since no precautions were taken to

ORIGIN FRONT

ORIGIN FRONT

ORIGIN FRONT 4 Rf VALUE 6

I@0 FIG. 2. Paper chromatograms of reaction mixtures contain-

ing SMX compared to a chromatogram of authentic H2ptCH,SMX. A, the complete reaction mixture (M) contained 2.0 nmol of [3-%]SMX, Hgpteroate synthase, 1.0 pmol of Mg*+, and 4.0 nmol of H2ptCH20PP in a total volume of 206 ~1. The control reaction mixture (0. . . .O) was incubated in the absence of H2ptCH20PP. Incubation was for 30 min at 37°C. B, the complete reaction mixture (M) contained HzptCHsOH pyrophosphokinase, 1.32 nmol of [2-‘4C]H2ptCH20H, 0.5 pmol of ATP, and 1.0 pmol of Mg*+ in a total volume of 160 ~1, and was incubated 30 min at 37°C before adding Hzpteroate synthase and 2.0 nmol of SMX (total volume, 200 ~1). The reaction mixture was then incubated an additional 30 min at 37’C. The control (0.. . .O) was incubated in the absence Hzpteroate synthase. C, the reaction mixtures were identical with those described in B except 2.0 nmol of [3-%I]SMX replaced nonradioactive SMX. D, authentic HzptCHzSMX, located by viewing under UV light (254 nm).

avoid oxidation during chromatography, the shoulder at an RF value of 0.84 probably represents [2-‘?]ptCH20PP.

Further evidence that the product at RF value of 0.24 contained both a pteridine moiety and sulfamethoxazole was obtained from a double label experiment with [2-r4C]- HzptCHzOPP and [3-35S]SMX. As seen in Fig. 2C, the complete reaction mixture shows a new peak at an RF value of 0.24 which accounts for 70% more radioactivity than the product synthesized from either radioactive substrate alone.

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The radioactive compound synthesized in the complete reaction mixture also migrates to the same position as authentic HzptCH$MX in several other solvents tested (see Table I). The enzymatic product fluoresced under UV light which is consistent with the behavior of HzptCH$SMX. As observed by Bock et al. (12), the protein content of the reaction mixture probably alters the RF value of SMX compared to the RF value of SMX when chromatographed alone.

Other sulfonamides were tested as substrates in assays similar to those described above. The product synthesized from H2ptCH20PP and [35S]SA migrates to an RF value of 0.06, whereas [35S]SA migrates to an RF value of 0.76. When STZ and [2-‘4C]H2ptCH20PP were the substrates, the radioactive product had an RF value of 0.07, whereas [2-35S]STZ migrated to an RF value of 0.70. The RF values of these products correspond to the RF values of authentic HzptCHzSA and H2ptCH2STZ, respectively.

Kinetics of HGteroate Synthase in the Presence of Sulfon- amides-[ carboxy-‘4C]p-AB incorporation into Hzpteroate was inhibited competitively by the sulfonamides tested and, a replot of the slopes versus the inhibitor concentrations indicated linear competitive inhibitions (11). The kinetic constants are shown in Table II. SMX was noncompetitive with HzptCHzOPP with an apparent KII value of 8.19 + 1.36 pM when the concentration of p-AB was 20 PM. Replots of both the slopes and the intercepts versus inhibitor concentration indicate linear noncompetitive inhibition.

As demonstrated above, SMX can replace p-AB as a substrate for Hzpteroate synthase. The product synthesized from [3-?S]SMX was chromatographed on paper and quantitated by analyzing for radioactivity a section of the chromatogram that was shown to include all the radioactivity associated with the product and none of the unreacted [3-35S]SMX. The kinetic constants for SMX are included in Table II. [3- 35S]SMX incorporation into product was competitively in-

hibited by p-AB with an apparent K,, value of 0.53 f 0.07 PM. Hzpteroate synthase in the presence of [35S]SA (18.6 Ci/ mol) or [35S]STZ (2.1 Ci/mol) was also studied. Since the products formed from these sulfonamides migrated only slightly when chromatographed on paper they were quantitated as described for Hzpteroate (5). Although the specific activity of the [35S]STZ was too low for accurate kinetic determinations, the apparent K,,, value appeared to be tl PM. Each sulfonamide was evaluated as a growth inhibitor of E. coli B and these results are included in Table II. The values reported here are consistent with previously published values (1).

Penetration Studies-The permeability of the cells to p- AB and sulfonamides was determined by measuring the cellular and extracellular concentrations of radioactivity following the incubation of cells in the presence of radioactive compounds. The results presented in Table III indicate that p-AB was concentrated no more than 3.4-fold when diffusion was studied for 30 min at 4°C. This type of experiment does not eliminate active transport as a means of uptake, nor does it necessarily mean that the cellular concentration indicated is actually the intracellular concentration.

Studies conducted with each of the sulfonamides indicated that the concentration factor (cellular concentration/Cfj) was never greater than 2.7 and generally was approximately 2.0 (Table III). The inclusion of 0.1% toluene to increase the cellular permeability did not lower the results with sulfanilamide, thus suggesting that the radiolabel was bound to cell components (42). [3-35S]SMX could be washed away from the cells with nonradioactive SMX. The diffusion of STZ, with a pK, of 7.1 (43), was studied at pH 6.0, pH 7.0, and pH 8.0, and no differences were seen in the concentration factor, indicating that the degree of ionization of this sulfonamide extracellularly was not a factor in its uptake. Incubating SA and SMX at 37°C for 5 min or 4°C for 30 min resulted in similar concen-

TABLE I Migration on TLCplates of substrates and products from reaction mixtures containing [3-3”S]SMX

Both reactions contained 8 nmol of [3-““SISMX, Hzpteroate synthase, 16 pmol of Tris-Cl, pH 8.3 (37”C), and 1.0 pmol of Mg*‘, in a total volume of 400 ~1. The complete reaction was incubated in the presence of 4.0 nmol of H2ptCHzOPP. Each chromatogram was spotted with 50 ~1 of the reaction mixtures.

R,

SMX (20 nmol) Control reaction Complete reaction H2ptCH2SMX (6 nmoll

” A = ultraviolet absorbant. b F = ultraviolet fluorescent.

5% Na2HP04 0.21% LiCl 3% NH&l

WI UV254 [““Sl uvm ?Sl uv254 0.86 (A)” 0.46 (A) 0.53 (A)

0.70 0.70 (A) 0.71 0.69 (A) 0.63 0.69 (A) 0.23 0.25 (F)* 0.40 0.41 (F) 0.13 0.12 (F)

0.28 (F) 0.38 (F) 0.12 (F)

TABLE II

Kinetic constants of sulfonamides as substrates and inhibitors of H2pteroate synthase and as growth inhibitors of E. coli B Htpteroate synthase

Compound Substrate activity appar- Substrate V,,,, as % Inhibitor activity apparent ent K,,” Vm P-AB K,s”

Growth inhibition EDm

PM p-AB 0.57 + 0.05” (100) Sulfathiazole <l.O -75 Sulfamethoxazole 0.12 + 0.02 75 Sulfanilamide 4.8 + 1.4 30

“K,,, f S.E. b Inhibition constant determined from slopes of l/[u] uersus l/[s] plots kS.E. ’ Ref. 25. ‘Value determined using [3-““Sjsulfamethoxazole as substrate. ’ Values determined using [carboxy-‘%]p-AB as substrate. ‘Concentration required to inhibit the growth rate by 50% f S.E.

P PM 0.53 + 0.07” -

0.035 + 0.003’ 3.8 + 0.3’ 0.13 f 0.017’ 1.5 + 0.36

5.7 f 0.5’ 1156.0 f 68

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TABLE III

Summary of diffusion studies on p-AB and the sulfonamides A B Average of

Experimental condi- the range of tions pH 7.0 Cellular con- concentra-

c;’ range centration tion factor, range B/A

PM Pf p-AB 30 min, 4’C 0.026-3.89 0.065-13.3 2.8 SMX 30 min, 4°C 0.302-6.38 0.475-7.75 1.49 SMX 5 min, 37°C 0.317-6.47 0.40-7.43 1.16 STZ 30 min, 4°C 2.5-49 5.3-104 2.12 SA 30 min, 4°C 6-587 15-1350 2.2 SAh 30 min, 4’C 300 597 1.88 SA 5 min, 37°C 5.9-609 16-1253 2.25

n The final concentration of solute in the presence of cells, ’ Measured in the presence of 0.1% toluene.

tration factors, which suggests that these sulfonamides were not actively transported. The data indicate that the sulfonamides diffuse into E. coli and that the concentration associated with the cells is equivalent to the extracellular concentration. SMX and SA were not concentrated at 37”C, therefore, if they are metabolized, the metabolites diffuse similarly to the sulfonamides.

In order to determine whether sulfonamides are actively transported, the uptake of [35S]SA (9.1 Ci/mol) by E. coli ML30 was studied. The radioactivity retained by the filter in the presence or absence of the cells at zero incubation time was 50 to 70 cpm. If 100% of 0.5 PM [35S]SA was transported, an additional 1000 cpm would be retained. There were no additional counts associated with the cells over the range of 0.5 to 1000 pM of [35S]SA incubated at 31°C for up to 3 min. As a control for this methodology, the energy-dependent uptake of L-[U-‘4C]leucine (308 Ci/mol) was demonstrated. E. coli ML30 cells transported 65% of 0.5 pM radioactive leucine added to the cells over a 2-min time course of incubation at 25°C.

In Viva Synthesis of ptCH&MX-Bock et al. (12) found materials chromatographically similar to ptCHzSMX and HsptCHzSMX in the extracellular medium of E. coli grown with SMX, but no attempts were made to isolate, identify, or quantitate these materials. In our study, a large culture of E. coli was grown with [3-35S]SMX anaerobically (to prevent oxidative cleavage of the product) in Tris medium. Glucose minimal medium was unacceptable because the product breaks down to a compound that absorbs at 420 nm. Following incubation, the cells were collected and a chromatogram of a 400~~1 sample of the culture supernatant indicated that 29% of the [3-35S]SMX had been metabolized to two products which migrated to the positions of authentic ptCHSMX and HzptCH2SMX.

To isolate the products, the culture supernatant fluid containing 137 nmol of products was applied to a DE52 column. Application of the buffer containing 1.0 M KC1 eluted a symmetrical peak of radioactivity which contained both the radio- labeled products (103 nmol) and was free of [3-35S]SMX. These fractions were pooled and were concentrated and the material was purified as described under “Materials and Methods.” The material recovered from charcoal (73 nmol, 90% of the applied amount) showed one symmetrical 35S peak on paper chromatograms and a single spot on TLC plates (5% Na2P04), at RF values corresponding to ptCH$SMX. It is likely that the handling of the biosynthesized H*ptCH$SMX during the purification resulted in its oxidation to ptCH$SMX. The UV spectrum of the [3-35S]ptCH2SMX, shown in Fig. 3, is similar to that of authentic ptCH&MX. The concentration of the [3-35S]ptCH2SMX based on the extinction value at 365 nm (8700) was 1.4 times higher than the specific radiolabel

concentration. This difference may be due to the contamination with other pteridines.

The identification of the [3-35S]ptCH2SMX was confirmed by employing reverse phase high pressure liquid chromatography techniques. After initiation of the solvent gradient authentic ptCH$MX was eluted at 54 min and [3-35S]SMX at 40 min. The elution profiles of authentic ptCH2SMX and biosynthesized [3-35S]ptCHzSMX are identical (Fig. 4). The radiolabel (94%) was recovered in a peak that directly overlays the ultraviolet absorbing peak of authentic ptCH2SMX at 54- min elution time. The remaining 35S label elutes at 40 min which probably represents slight breakdown of the [3-35S]- ptCHzSMX to free [3-35S]SMX.

In Vivo Concentration of HzptCHd3MX-Since we confirmed the in uivo synthesis of HzptCHzSMX by E. coli B, it was necessary to determine the cellular concentration of the product in order to evaluate the physiological significance of the pterin-sulfonamides. The cellular concentration of 35S label was measured during the aerobic growth of E. coli B because of the relatively short duration of the experiment and the ease of sampling every 30 min. Since the amount of 35S

08r 06 -

0.0 L ’ I I I I I 1 I

240 280 320 360 400 WAVELENGTH, nm

FIG. 3. Ultraviolet absorption spectra of authentic ptCH&MX (M) and of [3-%]ptCH2SMX M) isolated from E. coli B. Spectra were recorded in 0.1 N NaOH at 20 PM (~26~ 32,400) pterin. The radiolabel concentration of [3-35S]ptCHzSMX was 14 WM.

1

I I I 1 I , , 1 I I I I

0 5 IO 15 20 25 30 35 40 45 50 55 60 65 ELUTION TIME,MINUTES

FIG. 4. High performance liquid chromatography elution profile of authentic ptCH%SMX and of [3-%]ptCHzSMX. A sample (55 ~1) containing 5.3 nmol of [3-?S]ptCH2SMX (by radiolabel) was combined with 10 ~1 containing 29 nmol of ptCHklMX and then separated by the use of high performance liquid chromatography. The arrows denote the positions of [3-%]SMX and of authentic ptCH2SMX as determined in separate experiments. The solid line is the AZM tracing and the radioactivity is denoted by x.

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found associated with the cells was 55 pmol, this experiment was repeated several times with the highest specific activity [3-35S]SMX available (28 Ci/mol). Although this procedure measures both the product and the unreacted [3-35S]SMX found intracellularly, several separate experiments indicated that the intracellular concentration of total 35S label was 52 w. In the experiment presented in Fig. 5, the concentration was 1.06 f 0.6 pM, where the large standard error reflects the inherent inaccuracy of these small numbers.

The rate of the appearance of the products extracellularly was monitored from identical cultures. The concentration of the extracellular products presented in Fig. 5 represents both oxidized and reduced ptCHSMX, separated from [3-35S]SMX by paper chromatography and quantitated as described. At an Am of 0.5, the extracellular concentration of products was 0.25 PM, representing a 40% conversion of the [3-35S]SMX to products. If the biosynthesized products that were found in the culture supematant had remained inside the cell and did not diffuse out, the steady state intracellular concentration would have been 911 f 26 pM. This value is calculated from the regression analysis of a plot of the total volume of intracellular water calculated from the optical density versus the total nanomoles of the product found outside the cell. These experiments show that >99% of the ptCH&SMX biosynthe-

0.1 0.2 0.3 0.4 0.5 0.6 ABSORBANCE (600 nm)

FIG. 5. Total amount of cellular and extracellular [3-?!+ ptCHzSMX with the growth of E. coli B. See “Materials and Methods.”

sized by E. coli B diffused into the extracellular media and was not concentrated in the cell.

Growth Inhibition Studies-Because H2ptCH2SMX is freely diffusible, monitoring the growth rate of E. coli B in the presence of 2 pM HzptCHzSMX extracellularly should elucidate its effects on growth. The anaerobic growth rate (Iz f S.E.) of a culture containing 2 FM H2ptCH2SMX (K = 0.375 + 0.002) was inhibited by 11% compared to the control growth rate (k = 0.422 + 0.003). The aerobic growth of E. coli B (K = 0.946 + 0.009) was inhibited by 12% at 34 pM ptCH$MX (Iz = 0.833 f 0.005). In both cases, the inhibition could be accounted for by the slight contamination of the pterin-sul- famethoxazoles by free SMX.

In order to investigate the inherent effects of the pterin- sulfonamides, the authentic aromatic and reduced compounds were tested as inhibitors of various folate biosynthetic and cofactor-utilizing enzymes and the results are presented in Table IV. The substrate concentrations were within 4-fold higher than their apparent K,,, values. The variation in the highest concentration of the compounds tested is directly related to the poor solubility of these compounds at the pH of the assay described.

H2ptCH2SMX and HzptCHzSTZ are moderately inhibitory to the folate biosynthetic enzymes tested, with IS0 values ranging from 2 to 150 PM. These compounds are less inhibitory than HPpteroate (1% value of 55 pM) against GTP cyclohydrolase (44). They were most potent as product inhibitors of Hzpteroate synthase. The inhibition by HzptCHzSTZ was competitive with H2ptCH20PP with an apparent K,, of 1.33 + 0.11 pM; a value similar to the apparent K,, value (0.6 FM) for Hzpteroate. H2ptCH2STZ was noncompetitive with p-AB, with an apparent Kit of 12.5 + 3.8 pM (H*pteroate apparent K,, = 9.5 rt 1.7 PM). Since the 150 values are similar for HzptCHzSTZ and H2ptCH2SMX against Hzpteroate synthase, it would be consistent for their K, values to be much the same also. H2ptCH2STZ and H2ptCH2SMX also inhibit Hzfolate synthetase by 50% at 18 PM and 9.1 PM, respectively. The free sulfonamides at millimolar concentrations have no effect on this enzyme.

The aromatic and 7,8-dihydropterin-sulfonamides were in- active or only weakly inhibitory against the activity of the following Hlfolate cofactor-utilizing enzymes: serine hydroxymethyltransferase (EC 2.1.2.1), thymidylate synthetase (EC 2.1.1.45), N5,N’o-methenyl-Hlfolate cyclohydrolase (EC 3.5.4.9), and N5,N’o-methylene-H4folate dehydrogenase (EC 1.5.1.5).

DISCUSSION

Although previous reporters have indicated that sulfonamides are metabolized in the presence of Hzpteroate synthase,

TABLE IV

Inhibition bypterin-sulfonamide adducts of folate enzymes isolated from E. coli B

Enzyme ptCHSA HnptCHSA ptCH>STZ HlptCH,STZ ptCHzSMX %i !.ud 81 PM %i PM %i PM %i pM

GTP cyclohydrolase 0 51 0 51 0 100 50 90 0 100 H2ptCH20H pyrophosphokinase ND” 0 48 10 148 50 158 23 363 H?pteroate synthase 50 207 50 148 23 10’ 50 2.2 34 62b Hsfolate synthetase 0 100 0 35 0 148 50 18 0 154 Hzfolate reductase 0 24 0 35 0 4.7 ND 0 123 Serine hydroxymethyltransferase 61 140 41 140 7 118 16 57 0 123 Thymidylate synthetase 0 112 0 120 18 74 18 71 0 123 N”,N”-methylene-H4folate dehy- 0 56 0 65 0 22 22 22 0 123

drogenase N”,N’“-methenyl-H4folate cycle- 0 24 0 2.4 0 28 0 28 0 62

hydrolase

n ND, not determined. * These values determined at 20 pM HzptCHzOPP.

HrptCHSMX

%i PM 50 150 50 46 50 3.0 50 9.1

0 148 0 148 0 148 0 79

29 74

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neither the detailed kinetics of the reaction, nor its role in growth inhibition by sulfonamides have been investigated. We found that the apparent K,,, values for p-AB and for each sulfonamide as a substrate for Hzpteroate synthase are equivalent to their apparent K,, values as competitive inhibitors, a characteristic of competitive inhibitors which are alternate substrates of the physiological substrate (11). The kinetic values presented in this paper show that SMX and probably STZ bind more tightly than doesp-AB to Htpteroate synthase and that the reactions catalyzed proceed at similar rates.

As a result, the rate of HzptCHzOPP utilization will be similar in the presence and absence of these sulfonamides. Accumulation of the pterin substrate within the cell will thus not occur, as it most probably would if sulfonamides were not alternate substrates. Although SMX has been shown to be a noncompetitive inhibitor with respect to HzptCHzOPP, under certain conditions accumulation of the pterin substrate could nevertheless decrease the inhibition by SMX. Therefore, by preventing the accumulation of HzptCHzOPP, the ability of sulfonamides to act as alternate substrates of Hzpteroate synthase contributes to their effectiveness as inhibitors of folate biosynthesis, and thus, of growth.

There have been several previous reports on the uptake of sulfonamides by various microorganisms. Although Reddy et al. (45) has reported that sulfadiazine is actively transported by E. coli, Buttner and Biittner (42) have shown sulfadiazine to be concentrated no more than 1.5-fold by E. coli. STZ was not concentrated by various sensitive and sulfonamide resist- ant Staphylococci strains (46). In the latter two studies, the sulfonamide could easily be washed out of the cells.

The results presented in this paper (Table III) show that SA, SMX, and STZ diffuse into E. coli B. The results of the uptake studies are supported by the experiment determining the steady state intracellular concentration of 35S radiolabel to be 52 pM. This finding is totally consistent with the fact that SMX diffuses into E. coli at micromolar concentrations, and, in addition, establishes the upper limit for accumulation of the H2ptCHzSMX product.

Based on the kinetic data, the intracellular concentrations of SMX predicted by the penetration studies are sufficient to inhibit Hzpteroate synthase to the extent demanded by the observed inhibition of growth, if the intracellular concentration of p-AB is 55 ,uM (Km = 0.5 PM).

Among the enzymes tested, the most potent inhibition by the Hzpterin-sulfonamides was against Hzpteroate synthase. Product inhibition patterns with Hzpteroate synthase indicate an ordered reaction sequence where H2ptCH20PP is the fist substrate to bind and Hzpteroate is the second product re- leased (22). The Hzpterin-sulfonamides are competitive inhibitors with HzptCHpOPP, and therefore they are acting as Hzpteroate analogs and are binding to the same form of the enzyme. The Hzpterin-sulfonamides were also found to be inhibitors of the next enzyme of the folate biosynthesis sequence, dihydrofolate synthetase. To evaluate the effects of these actions on the overall inhibition of folate biosynthesis, a rate expression for the operation of the sequence was de- rived, similar to and based on that for a two-step product- inhibited sequence (47). Hzpterin-sulfonamide was assumed to diffuse from the cell, in accordance with the data presented in this paper, with the steady state concentration determined by the choice of value of the first order rate constant for diffusion.

Steady state reaction velocities of the sequence were calculated using the kinetic parameters for substrates and inhibitors reported in this paper and in Refs. 25 and 28. It was assumed thatp-AB and H2ptCH20PP were present at concentrations equal to their K,,, values. Hzpterin-sulfonamides were

assumed to be either competitive or noncompetitive with H2pteroate in the inhibition of dihydrofolate synthetase. None of these assumptions had any substantial effect upon the conclusions. The calculations showed that inhibition of Hzpteroate synthase and H2folate synthetase by Hzpterin- sulfonamides could be significant only when the product accumulated in the cell to a considerably higher level than that observed experimentally. For example, with SMX present at a concentration sufficient to inhibit the pathway velocity by 40% due to competition with p-AB, a further 2-fold reduction in pathway velocity due to inhibition of Hzpteroate synthase and Hzfolate synthetase by H2ptCH2SMX occurred only when the product accumulated to a concentration of 8 pM. When accumulation was limited to 2 p, product inhibition by HzptCHzSMX increased the calculated inhibition of pathway velocity from 40 to 49%. Even this increase is less than expected from the effect on the enzyme in isolation, because of the associated reduction in product inhibition by Ha- pteroate. Inhibition of Hzfolate synthetase by HnptCHz- SMX further increased the level of inhibition by less than 1%. The negligible contribution of this inhibition results from the fact that it is readily overcome by a slight increase in Hzpteroate concentration, balanced by a similarly small decrease in H2ptCH2SMX. Calculations were made assuming a wide range of intracellular concentrations of SMX, and the results described above are typical of all. The conclusions drawn from the calculations are supported by the observed lack of inhibition of growth of E. coli by exogenously supplied H2ptCH2SMX. We therefore conclude that inhibition by H2pterin-sulfonamides of Hz-pteroate synthase, or of the other folate enzymes tested, is not physiologically significant. The use of sulfonamides as alternate substrates by H;?pteroate synthase contributes to growth inhibition only through preventing the accumulation of HaptCHaOPP which would otherwise occur. Competition with p-AB is thus the primary mode of action of sulfonamides. The consequent reduction in the rate of Hzpteroate synthesis decreases the concentration of H4folate cofactors in the cell, reducing the rate of synthesis of the products of the pathway, thus reducing the cell growth rate.

Acknowledgments-We are grateful to Dr. Paul H. Ray for his advice and instruction in the cellular permeability studies. We would like to thank Dr. Jon C. Nixon for his assistance in performing the high performance liquid chromatography techniques and helpful dis- cussions. We express our appreciation to Inderjit K. Dev, Pal.1 Novak, and Alice L. Warskow for their technical assistance.

1. 2.

3.

4. 5. 6.

7.

8.

9.

10.

11.

REFERENCES

Brown, G. M. (1962) J. Biol. Chem. 237.536-540 Shiota, T., Disraely, M. N., and McCann, M. P. (1964) J. Biol.

Chem. 239,2259-2266 Shiota, T., Jackson, R., and Baugh, C. M. (1970) in Chemistry

ancl Biology of Pteridines (Iwai, K., Goto, M., Akino, M. and Iwanami, Y., eds) pp. 265-279, International Printing Co., To- kyo

Ortiz, P. J., and Hotchkiss, R. D. (1966) Biochemistry 5,67-74 Ferone, R. (1973) J. Protozool. 20,459-464 Ho, R. I., Corman, L., Morse, S. A., and Schneider, H. (1975)

Antimicrob. Agents Chemother. 7,758-763 Pelczar. M. J.. Jr.. and Reid. R. D. (1965) Microbiolozv. DD. 343-

344, McGraw-Hill, New York -- __

Mahler, H. R., and Cordes, E. H. (1966) Biological Chemistry, p. 257, Harper and Row, New York

Davis, B. D., Dulbecco, R., Eisen, H. N., Ginsberg, H. S., and Barrywood, W., Jr. (1968) Microbiology, pp. 302-311, Harper and Row, New York

Lehninger, A. L. (1970) Biochemistry, p. 570, Worth Publishers, Inc., New York

Segel, I. H. (1975) Enzyme Kinetics, pp. 100-159, John Wiley & Sons, New York

by guest on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12.

13.

14.

15. 16. 17.

18.

19.

20.

Bock, L., Miller, G. H., Schaper, K. J., and Seydel, J. K. (1974) J. 30. Burchall, J. J., and Hitchings, G. H. (1965) Mol. Pharmacol. 1, Med. Chem. 17,23-28 126-136

Swedberg, G., Castensson, S., and Skold, 0. (1979) J. Bacterial. 31. Taylor, R. T., and Weissbach, H. (1965) Anal. Biochem. 13,80- 137, 129-136 84

Taylor, E. C., Portnoy, R. C., Hochstetler, D. C., and Kobayaski, 32. Harvey, R. J. (1973) J. Bacterial. 114, 309-322 T. (1975) J. Org. Chem. 40,2347-2351 33. Scrimgeour, K. G., and Huennekens, F. M. (1963) Methods En-

Forrest, H. S., and Walker, J. (1949) J. Chem. Sot. 2002-2007 zymol. 6,368-372 Martin, G. J., and Avakian, S. (1949) U. S. Patent 2,476,557 34. Dev, I. K., and Harvey, R. J. (1978) J. Biol. Chem. 253, 4245- Sato, H., Nakajima, M., and Tanaka, H. (1951) J. Chem. Sot. 4253

Jpn. 72,866-868 35. Greenberg, D. M. (1963) Methods Enzymol. 6, 386-387 Roberts, E. C., and Shealy, Y. F. (1974) J. Heterocycl. Chem. 11, 36. Winkler, H. H., and Wilson, T. H. (1966) J. Biol. Chem. 241,

547-550 2200-2211 Bieri, J. H., and Viscontini, M. (1973) Helu. Chim. Acta 56,2905- 37. Kaback, H. R. (1974) Methods Enzymol. 31,698-709

2911 38. Black, S. H., and Gerhardt, P. (1961) J. Bacterial. 82, 743-749 Blakley, R. L. (1960) Nature 118,231-232

21. Blaklev. R. L. (1969) The Biochemistrv of Folic Acid and Related Ptehhines pp. 58-98, John Wiley & Sons, Inc., New York

22. Friedkin, M. (1963) Methods Enzymol. 6, 124-129 23. Rabinowitz, J. C. (1963) Methods Enzymol. 6, 814-815 24. Baugh, C. M., and Shaw, E. (1964) J. Org. Chem. 29,3610-3612 25. Ferone, R., and Webb, S. R. (1975) in Chemistry and Biology of

Pteridines (Pfleiderer, W., ed) pp. 61-71, Walter de Gruyter and Co., Berlin


26. Brown, G. M. (1971) Methods Enzymol. MB, 761-765 27. Richey, D. P., and Brown, G. M. (1969) J. Biol. Chem. 244,1582-

1592 28. Webb, S. R., and Ferone, R. (1976) Biochim. Biophys. Acta 422,

419-426 29. Baccanari, D., Phillips, A., Smith, S., Sinski, D., and Burchall, J.

(1975) Biochemistry 14,5267-5273

39. Pattishall, K. H., Acar, J., Burchall, J. J., Goldstein, F. W., and Harvey, R. J. (1977) J. Biol. Chem. 252,2319-2323

40. Cleland, W. W. (1963) Nature 198,463-465 41. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. Biol. Chem. 193,265-275 42. Buttner, D., and Biittner, H. (1972) Chemotherapy 17,309-319 43. Bell, P. H., and Roblin, R. 0. (1942) J. Am. Chem. Sot. 64,2905-

2917 44. Nixon, J. C., Butz, R. F., Nichol, C. A., Dev, I. K., and Harvey, R.

J. (1975) Biophys. J.. 15, lla 45. Reddy, M. S., Williams, F. D., and Reinbold, G. W. (1973) Anti-

microb. Agents Chemother. 4, 254-258 46. White, P. J., and Woods, D. D. (1965) J. Gen. Microbial. 40, 255-

271 47. Harvey, R. J. (1978) J. Theor. Biol. 74,411-437

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S Roland, R Ferone, R J Harvey, V L Styles and R W MorrisonEscherichia coli dihydropteroate synthase.

The characteristics and significance of sulfonamides as substrates for

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The Characteristics and Significance of Sulfonamides as Substrates

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