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PYRIMIDINE BIOSYNTHESIS IN ESCHERICHIA COLI* BY RICHARD A. YATESt AND ARTHUR B. PARDEE (From the Virus Laboratory, University of California, Berkeley, California) (Received for publication, November 21, 1955) The recent great interest in the formation and function of nucleic acids has caused a similar interest in the synthesis of such component parts of nucleic acid as the pyrimidines. Implicated in pyrimidine biosynthesis have been the compounds aspartic acid (1, 2), ureidosuccinic acid (US) (l-5), dihydroorotic acid (DHO) (6), erotic acid (OA) (4-S), 5-phosphori- bosylpyrophosphate (PRPP) (9), orotidine-5’-phosphate (0-5’-P) (10, ll), uridined’-phosphate (U-5’-P) (10, 11), and cytidine triphosphate (CTP) (1% A composite scheme of pyrimidine biosynthesis drawn from the above references can be summarized in the accompanying diagram, all steps with solid arrows representing known enzymatic reactions. Aspartic acid I f2H (A) us &He0 03 DHO \ \ (C) Carbamyl phosphate OA . PRPP o-5’-P -coz - U-5/-P 33 CTP I / / i J/ nucleic acid The enzyme found for step (A) has been named ureidosuccinic acid synthetase by Jones et al. (l), and the enzymes for steps (B) and (C) are named, respectively, dihydroorotase (5) and dihydroorotic acid dehydro- genase(6) by Lieberman and Kornberg. The study of pyrimidine biosynthesis by Lieberman, Kornberg, and Simms has been outstanding in revealing enzymatically the whole pathway from step (B) on. One limitation to the conclusions drawn from their earlier work (5, 6) concerning the steps from US through OA, however, is that organisms (Zymobucterium oroticum (6) and some corynebacteria (13)) grown on OA media were used as source material for enzyme studies. Un- * Presented in part at the Pacific Slope Biochemical Conference, 121st meeting of the American Association for the Advancement of Science, December 30,1954. Supported, in part, by research grants from the Lederle Laboratories Division, American Cyanamid Company, and the Rockefeller Foundation. t Abraham Rosenberg Research Fellow during the course of this work. 743 by guest on June 22, 2020 http://www.jbc.org/ Downloaded from

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Page 1: PYRIMIDINE BIOSYNTHESIS IN ESCHERICHIA COLI* · PYRIMIDINE BIOSYNTHESIS IN ESCHERICHIA COLI* BY RICHARD A. YATESt AND ARTHUR B. PARDEE (From the Virus Laboratory, University of California,

PYRIMIDINE BIOSYNTHESIS IN ESCHERICHIA COLI*

BY RICHARD A. YATESt AND ARTHUR B. PARDEE

(From the Virus Laboratory, University of California, Berkeley, California)

(Received for publication, November 21, 1955)

The recent great interest in the formation and function of nucleic acids has caused a similar interest in the synthesis of such component parts of nucleic acid as the pyrimidines. Implicated in pyrimidine biosynthesis have been the compounds aspartic acid (1, 2), ureidosuccinic acid (US) (l-5), dihydroorotic acid (DHO) (6), erotic acid (OA) (4-S), 5-phosphori- bosylpyrophosphate (PRPP) (9), orotidine-5’-phosphate (0-5’-P) (10, ll), uridined’-phosphate (U-5’-P) (10, 11) , and cytidine triphosphate (CTP) (1%

A composite scheme of pyrimidine biosynthesis drawn from the above references can be summarized in the accompanying diagram, all steps with solid arrows representing known enzymatic reactions.

Aspartic acid

I

f2H (A) us ’

&He0

03 ’ DHO \

\

(C) Carbamyl phosphate

OA . PRPP

’ o-5’-P -coz

- U-5/-P 33 CTP

I /

/

i J/ nucleic acid

The enzyme found for step (A) has been named ureidosuccinic acid synthetase by Jones et al. (l), and the enzymes for steps (B) and (C) are named, respectively, dihydroorotase (5) and dihydroorotic acid dehydro- genase (6) by Lieberman and Kornberg.

The study of pyrimidine biosynthesis by Lieberman, Kornberg, and Simms has been outstanding in revealing enzymatically the whole pathway from step (B) on. One limitation to the conclusions drawn from their earlier work (5, 6) concerning the steps from US through OA, however, is that organisms (Zymobucterium oroticum (6) and some corynebacteria (13)) grown on OA media were used as source material for enzyme studies. Un-

* Presented in part at the Pacific Slope Biochemical Conference, 121st meeting of the American Association for the Advancement of Science, December 30,1954.

Supported, in part, by research grants from the Lederle Laboratories Division, American Cyanamid Company, and the Rockefeller Foundation.

t Abraham Rosenberg Research Fellow during the course of this work. 743

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744 PYRIMIDINE BIOSYNTHESIS IN E. COLI

der such growth conditions enzymes able to degrade OA may be formed adaptively to provide energy and constituents for cell growth, and such enzymes may be unrelated to normal pyrimidine biosynthesis.

It was desired here to determine both qualitatively and quantitatively whether the early steps in the biosynthesis proceed as shown above or by an alternative mechanism as suggested by the work of Mitchell et al. with Neurospora crassa mutants (7). Escherichia coli, strain B, and two pyrim- idine-requiring E. coli mutants were used for this study, grown under con- tions for which all enzymes studied would presumably be present only to synthesize pyrimidines.

Materials and Methods

Uracil, OA, and m-aspartic acid-4-C14 were purchased from the Cali- fornia Foundation for Biochemical Research. nL-Ureidosuccinic acid pre- pared by the method of Nyc and Mitchell (14) had a melting point at 176.3” (decomposed). A mixed melting point with DL-US obtained from Dr. H. A. Barker showed no depression.

L-Dihydroorotic acid was prepared by the method of Miller et al. (15) from carbethoxy-L-asparagine (16). Twice recrystallized from water, the product melted at 173” (uncorrected) and had the following composition: CsH,04Nz calculated, C 37.98, H 3.93, N 17.71; found, C 38.17, H 3.97, N 17.60 per cent.

A culture of 2. oroticum, isolated by Dr. A. Kornberg (17) and charac- terized by Wachsman and Barker (18), was kindly provided by Dr. H. A. Barker. E. coli mutant 550-460 (ATCC 11548) and mutant 6386 (iso- lated by Dr. B. D. Davis), both of which require pyrimidines, were present in the collection of this Laboratory.

Cl4 was determined in a gas flow Geiger-Miiller counter with platinum plates.

Bacterial Medium and Growth Conditions-E. coli strains were grown with aeration by swirling at 37” in a minimal medium of the following composition in gm. per liter: 7 K2HP04, 2 KHZPOI, 0.5 sodium citrate .- 5Hz0, 0.1 MgS04v7Hz0, 1 (NH4)&04, 6.3 glycerol (or 2.5 glucose) (19). In some cases the medium was supplemented with 2 X 10e4 M uracil or OA as growth factor.

2. oroticum cultures were grown on OA medium, harvested, suspended in the presence of OA in vacua, washed, and suspended in ice water as de- scribed by Lieberman and Kornberg (6). Cultures were similarly grown and prepared as above in the absence of OA, glucose replacing OA as a carbon source. Extracts were prepared by treatment in a 10 kc. Ray- theon magnetostriction (sonic) oscillator for 20 minutes in the presence of 200 mg. of glass powder. The extracts were centrifuged for 15 minutes at

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R. A. YATES AND A. B. PARDEE 745

10,000 X g, and protein determinations on the supernatant solution were made by the Folin method (20) standardized against the bacterial extract by biuret assays. All preparation and storage of the cell-free extracts were carried out near 0”. Storage for long periods of time was at -10”.

Cell-free extracts of E. c&i were also prepared in the sonic oscillator. For comparison with 2. oroticum, the conditions used were identical with those reported above. For other experiments extracts were prepared by sonic oscillation of cells suspended in ice water at a concentration of 2.5 X lOlo cells per ml. for 2.0 minutes. The extracts were centrifuged for 5 minutes at 9000 X g to remove whole cells and cellular debris.

Bacterial Counting Methods-The number of E. coli cells per ml. of cul- ture was determined either with use of a Petroff-Hausser bacterial counter or by means of turbidity readings (corrected to actual bacterial count) in a Klett-Summerson photometer with a green filter (No. 54). 1 Klett unit is equivalent to 0.9 X 10’ cells per ml.

Assay for Pyrimidine Intermediates-As a qualitative and semiquantita- tive test for the presence of the various intermediates, paper chromatog- raphy was carried out. In a butanol-ethanol-formic acid-water system (by volume 5:3:2: 1) (21), for OA RF = 0.42, for DHO RF = 0.45, and for US RF = 0.53 in descending chromatograms. OA spots were detected by ultraviolet absorption and US and DHO spots by color procedures by using the Ehrlich reagent (22).

Quantitative determinations of OA were made spectrophotometrically at various pH values (23). For the most accurate results, reaction and control samples were passed through Dowex 50 (acid form) columns to re- move amine-containing contaminants and were read at 270, 280, and 290 mp. Orotic acid can be quantitatively recovered in the eluate from the column.

Ureidosuccinate was assayed quantitatively by the calorimetric method of Koritz and Cohen (24). Dowex 50 columns were used as above to re- move traces of amine-containing ureido compounds.

Dihydroorotate was assayed by taking advantage of the large loss of optical density (maximal at 230 mp) found to occur upon alkaline hy- drolysis of DHO to US. The rate of hydrolysis is first order with respect to DHO at any given alkali concentration (25). In general, 1.5 ml. of 1 N sodium hydroxide were added to 1.5 ml. of sample in silica Beckman cells at room temperature, mixed rapidly, and read at 240 rnp for 15 min- utes at timed intervals. The final equilibrium optical density was sub- tracted from all values, and the semilog plot of corrected optical density was extrapolated linearly to zero time. No substances in the assay mix- tures except DHO changed their optical density significantly in 15 min- utes, Both optical extinction coefficient (E) and hydrolysis rate constant

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746 PYRIMIDINE BIOSYNTHESIS IN E. COLI

(k,) vary as alkali concentration is changed, but both reach a plateau and remain fairly constant over an alkali concentration range of 0.2 to 0.8 M

NaOH, making the assay essentially independent of buffer concentration and of acid or CO2 produced by the bacteria. In this plateau range, at 25”, e = 6.15 X 1Oa mole-‘, and k, = 0.17 min.-‘. A mechanism for the reaction will be proposed elsewhere.

EXPERIMENTAL

Study of E. coli Mutant-s-In thii study of pyrimidine biosynthesis in E. coli, two pyrimidine-requiring mutants, as well as wild type E. coli, strain B, were used. An effort was made to characterize the point at which biosynthesis was blocked and to determine the rates of utilization or pro- duction of pyrimidines or intermediates. These data were later correlated with those obtained from enzyme studies with cell-free extracts.

Preliminary studies showed that mutant 550-460 grew in minimal media containing uracil, uridine, or cytosine, but not thymine, OA, or DHO as growth factors. In the absence of growth factor, this mutant produced large amounts of erotic acid, which could be precipitated from concen- trated bacterial supernatant solutions as the potassium salt (26,7). After purification by charcoal, conversion to the acid form, and recrystalliza- tion from water, the bacterial product and commercial erotic acid had identical absorption spectra at all pH values (23), identical titration pK, values and equivalent weight, identical melting points, showed no depres- sion of a mixed melting point, and both behaved identically on chromato- grams (see “Materials and methods”).

Although not isolated, US and DHO have been shown to be present along with OA in minimal supernatant solutions of this organism. As shown in Fig. 1, the typical ratio of OA: DHO:US was about 15: 1: 6. The rate of production of OA + DHO + US varied slightly with condi- tions of growth, but was about 11 X 10-l’ mole per cell per hour in minimal media. In one division time (1.0 hour) 6.7 X 10-l’ mole per cell of uracil was taken up by the mutant and a similar amount of nucleic acid pyrim- idine was formed.

In contrast to mutant 550-460, mutant 6386 was observed to use OA as well as uracil for a growth factor, but not to use DHO or US. Several substrains of this mutant could be differentiated by their growth rates with OA. The division times observed were 1.1, 1.8, 3.3, 6.8, and 10 (or more) hours.

As expected, no OA was produced by suspensions of this mutant in minimal medium; however, DHO was present. The DHO was isolated from one minimal supernatant solution containing only 5 per cent the normal salt concentration by chromatography on Dowex 1 (formate), fol-

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R. A. YATES AND A. B. PARDEE 747

lowed by removal of cations by Dowex 50 (acid form) resin and vacuum concentration to dryness (6). After three recrystallizations from water, the bacterial product and synthetic n-dihydroorotic acid had the same melt- ing point (274” (decomposed)) with no depression of a mixed melting point, the same optical rotation, the same extinction coefficient and hydrolysis

-50---1250

o- I 2 3 4 HOURS

FIG. 1. Production of pyrimidine intermediates by E. coli mutants. See the text for details. The dotted lines are used for mutant 6386 and the solid lines for mutant 550-460. l , dihydroorotic acid production; W, ureidosuccinic acid production; 0, erotic acid production; A, total production of pyrimidine intermediates; + , cell concentration of mutant 6386 and of mutant 550-460.

rate constant in alkali, and identical chromatographic properties with the solvent mixture used above for OA.

As shown in Fig. 1, US as well as DHO was produced by this mutant when suspended in minimal medium. As with mutant 550-460, the rates of US and DHO production varied slightly with conditions used, but under the same conditions reported for mutant 550-460, mutant 6386 produced US and DHO in a ratio of 1.4: 1 and total production of 11 X 10-l’ mole per cell per hour. Also, in one division time (1 hour) 6.7 X 10-l’ mole

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748 PYRIMIDINE BIOSYNTHESIS IN E. COLI

per cell of added uracil or OA was consumed by the growing cells (Le., 1 pmole allowed production of 1.5 X lOlo cells).

Studies on Cell-Free Extracts-Z. oroticum cells were grown and cell-free extracts were prepared as described under “Materials and methods.” For the study of DHO oxidation, the reaction mixtures were read in a Beckman

0 20 40 60 MINUTES

FIQ. 2. Activities of dihydroorotase and of DHO dehydrogenase by extracts of 2. oroticum grown in the presence or absence of OA. For DHO oxidation, the reaction mixtures contained 0.033 M potassium phosphate, pH 6.5, 0.005 M MgC&, 0.005 M cysteine, pH 7.0,1.33 X lo-’ M DHO, and cell-free extract of 0.50 mg. of protein per ml. Total volume 3.0 ml. The substrate was added at zero minute to the reaction mixture and readings were made at 290 u against controls containing no substrate. OA values are magnified lo-fold. 0, 2. oroticum grown on OA enrichment media; 0, 2. oroticum grown on glucose media. For study of DHO hydrolysis, the reaction mixtures contained 0.033 M potassium phosphate, pH 6.5, 0.005 M MgCIt, 0.0017 M

Versene, 7 X 10-” M DHO, and cell-free extract of 0.50 mg. of protein per ml. Total volume 6.0 ml.; 37“; aliquots assayed at intervals for DHO as described under “Ma- terials and methods.” l , 2. oroticum grown on OA enrichment media; W, 2. orot- icum grown on glucose medium.

DU spectrophotometer at 290 rnE.1 against control samples lacking DHO. Fig. 2 shows that the initial rate of OA formation by extracts of 2. oroticum grown on OA medium was quite rapid, while extracts of 2. oroticum grown on glucose medium in the absence of OA had only about 5 per cent as much activity. As also demonstrated in Fig. 2, the activity of a second enzyme, dihydroorotase, was quite pronounced in extracts of 2. oroticum grown on OA, while extracts of the organism grown in the absence of OA were es- sentially inactive.

The extremely low activities of both these enzymes in 2. oroticum grown

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R. A. YATES AND A. B. PARDEE 749

on glucose compared to the activities of cells grown in the presence of OA (Fig. 2) indicate that the enzymes may have been formed adaptively on exposure of the cells to OA. The enzymes might have been formed only

! A , I J 0 40 80 120 160

MINUTES

FIG. 3. Interconversion of OA, DHO, and US by extracts of E. coli, strain B, and mutant 6386. Conditions, 0.033 M potassium phosphate, pH 6.5, 0.025 M MgC12, 0.001 MVersene, and 0.42 mg. of extract protein (2.4 X log cells) per ml. The total volume was 6.0 ml. At zero time, substrate (L-DHO or DL-US) was added to 0.001 M

concentration, mixed, and incubated at 37”. Samples were removed at intervals and assayed qualitatively and quantitatively for OA, DHO, and US as described in the text. The control samples lacked substrate. The values on the graph are concentrations in complete reaction mixtures minus values in control samples. OA values are magnified lo-fold. For E. coli, strain B, extract, DHO substrate plots are solid lines; 0 = DHO; A = OA; 0 = US. DL-US substrate plots are dash lines; l = US; q = DHO; A = OA. For mutant 6386 extract, DHO substrate plots are dotted lines; n = DHO; A = OA.

to use OA as a carbon source for metabolism and not to use it as a pyrim- idine precursor. The low but noticeable activity of the dehydrogenase in extracts of 2. oroticum grown on glucose media, however, might have been enough to account for the observed slow growth rate of the cells. Since quantitative determinations are difficult at the above low levels of activity, results obtained with cell-free extracts of the faster growing organism, E. coli, strain B, grown on minimal medium may be more pertinent.

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750 PYRIMIDINE BIOSYNTHESIS IN E. COLI

Fig. 3 presents results with extracts of E. COG, strain B, grown on mini- mal medium and extracts of mutant 6386 grown on this medium plus uracil. As can be seen, the E. coli extracts oxidized DHO to OA and converted DHO to US and US to DHO readily, with good stoichiometry, since only the L isomer of DL-US was used (6). The extracts of mutant 6386 con- verted DHO to US readily but did not form any OA, indicating that mu- tant strain 6386 lacks the enzyme dihydroorotic acid dehydrogenase. This finding fits well with the data obtained for the mutant in vivo.

As mentioned above, either DHO or US was converted to an equilibrium mixture of the two compounds by dihydroorotase. The equilibrium was determined over the range from pH 5.9 to 8.0 and found to be pH-de- pendent. At 37” the equilibrium constant [Kb = ((US=)(H+))/(DHO-)I had an average value of 1.4 X 10-S M at pH 6.0 and showed a slight de- crease with increasing pH over the range studied (at pH 8.0, K, = 0.6 X lo-‘j). This value for Ke compares fairly well with the value 1.5 X 10m6 M at pH 6.1 calculated from the experimental results of Lieberman and Kornberg (6).

Although the results from Fig. 3 indicate the presence of dihydroorotase and DHO dehydrogenase in E. co&i grown in minimal medium, the condi- tions used for the determinations were far from optimal. Fig. 4 shows that, for hydrolysis of DHO to US, the dihydroorotase has a pH optimum of 8.0. The shape of the dihydroorotase hydrolytic activity curve may be attributed to the titration of two groups in the active site of the enzyme (27) with pK values about 7.7 and 8.2 at 36”.

The optimal pH range for conversion of US to DHO is quite broad, with an optimum at about pH 6. The dihydroorotase activity for DHO forma- tion cannot be determined with any accuracy above pH 7.2, since the equilibrium ratio of US:DHO is quite unfavorable, but the pK of the active site of the enzyme is about 7.6, the same as that found above.

When enzyme reaction rates were studied as a function of substrate concentration and the results were plotted according to Lineweaver and Burk (28), the results in Fig. 5 were obtained. For conversion of US to DHO at pH 5.9, the Michaelis constant (K,) = 5.3 X lob4 M L-US, and maximal velocity (Vm) = 11 X lo-l6 mole per cell per hour, far above that required by cellular need for pyrimidines. Orotic acid is a competi- tive inhibitor in this system with an inhibition constant, Ki = 1.3 X 1OP M. It was found also that the activation energy for this conversion was 10,600 calories.

The conversion of DHO to US at pH 8.0 has K, = 3 X low4 M and vm = 18 X 1O-16 mole per cell per hour. The maximal velocity varies somewhat with different extracts and appears to require an unknown co- factor (not magnesium or calcium ions) at low extract concentrations. The activation energy for this conversion was found to be about 12,000 calories.

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R. A. YATES AND A. B. PARDEE 751

The dihydroorotic acid dehydrogenase from E. coli was also studied. The pH optimum for DHO oxidation is quite broad with a pK about 8.0,

5.0 6.0 7.0 8.0 9.0 PH

FIG. 4. Enzyme activities as a function of pH. All experiments were carried out at 37” with an E. coli, strain B, extract of 2.5 X 109 cells per ml. (0.46 mg. of protein per ml.). For ureidosuccinic acid synthetase ( l ) , the reaction mixtures contained 0.1 M potassium phosphate buffer, or 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer above pH 8, 0.01 M carbamyl phosphate, 2 X 10-4 M L-aspartic acid-4~04, and 5 X lo+ M magnesium chloride. The observed value at pH 6.2 was corrected to the calculated rate at saturating substrate concentrations, and other points were ad- justed proportionately. The curve is thus only approximately correct. With DHO as substrate, dihydroorotase (0) and dihydroorotic acid dehydrogenase (0) reaction mixtures contained 0.1 M Tris buffer, 0.061 M Versene, and 0.002 M DHO. The total volume was 5.0 ml. At timed intervals samples were passed rapidly through Dowex 50 (acid form) columns and assayed as described in the text. Initial reaction rates were calculated and plotted on the graph. With US as substrate, dihydroorotase (A) and dihydroorotic acid dehydrogenase (m) reaction mixtures contained 5 X 10m4 M Versene, 4 X 10-Q M DL-US, 0.1 M potassium phosphate buffer above pH 5.8, and acetic acid plus 0.1 M sodium acetate as buffer below pH 5.6. Other conditions as described above.

as shown in Fig. 4. At pH 7.25, Fig. 5 shows that K, = 4 X lo+ M

DHO and VWZ = 1.6 X lo-l6 mole per cell per hour, a rate more than fast enough to account for observed pyrimidine biosynthesis in whole cells. The Michaelis constant was determined by using an equilibrium mixture

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752 PYRIMIDINE BIOSYNTHESIS IN E. COLI

of US and DHO as substrate, as calculated from the pH and K,. The dihydroorotase in the extract, therefore, helped to keep the amount of DHO constant. From the relative shapes of the curves, the rate of pro- duction of OA from US is seen to be dependent on the rate of the dehydro- genase rather than of the dihydroorotase, as might be expected from the relative K, values.

.250 -

I , 0 1000 2000 3000

Ifi(LITERS / MOLE )

FIG. 5. Variations in enzyme reaction rates with substrate concentrations. Con- ditions were the same as those in Fig. 4 except as noted below. With US as substrate, dihydroorotase (0) reaction mixtures contained 0.1 M potassium phosphate, pH 6.0, 0.002 M Versene, and DL-US (calculated as L-US). The total volume was 5.0 ml. With DHO as substrate (0) the reaction mixtures contained 0.1 M Tris buffer, pH 7.8 (adjusted to 0.1 ionic strength with KCI), 0.001 M Versene, varied DHO, and extract of mutant 6386. Note scale of graph changed: Observed l/v and l/s both plotted as one-fourth actual values. For dihydroorotic acid dehydrogenase (A) the reaction mixture contained 0.1 M potassium phosphate, pH 7.4, mixture of L-DHO and DL-US in the ratio 1:50 (L-DHO:L-US of 1:25). Observed l/s and l/v both plotted as one- twentieth actual values.

A study was made also on the enzymatic synthesis of US from carbamyl phosphate and aspartate, first reported by Jones et al. (1). The method employed was to incubate E. coli extracts with carbamyl phosphate and CY4-aspartic acid under various conditions, then pass the reaction mixtures quantitatively through small columns of Dowex 50 (acid form) to adsorb the aspartic acid. Aliquots of the eluate were counted directly for Cl4 activity, or isotope dilution experiments with non-radioactive, synthetic IIL-US were performed.

Each method of assay has reasons to recommend it. Direct plating of

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R. A. YATES AND A. B. PARDEE 753

the eluate is the easier method, allows more sensitive determination of radioactivity, and assays total reaction rate (formation of US + DHO + OA) over a given interval, but must be corrected for the radioactivity from products of aspartic acid deamination. Although isotope dilution is a less sensitive method and only US is determined by it, the need for the above correction is avoided.

0 too 200 300 400 500 I/S (LITERS / MOLE)

FIG. 6. Variation of reaction rate ob ureidosuccinic acid synthetase with substrate concentration. The incubation mixture contained 0.1 M potassium phosphate, pH 6.5, 0.005 M MgCL, 0.010 M CP-nn-aspartic acid unless varied (results plotted as L-aspartic acid), 0.010 M carbamyl phosphate unless varied, and cell-free extract of 2.5 X 108 E. coli cells (0.44 mg. of protein). Total volume 1.0 ml. The reaction mixtures were incubated at 37” for 1.5 hours, rinsed through Dowex 50 (acid form) columns, plated, and counted directly in a gas flow Geiger-Mtiller counter. Cor- rections were made for control samples as described in the text. l , variable aspartic acid concentration; n , variable carbamyl phosphate concentration.

The non-enzymatic formation of US, first order with respect to both aspartate and carbamyl phosphate concentrations, also requires a correc- tion. At 0.03 M concentrations of each compound, US can be formed non- enzymatically at a rate of 0.48 pmole per ml. per hour, a rate faster than the enzymatic rate at the usual extract concentration of 2.5 X log cells per ml. To avoid this large non-enzymatic reaction, enzyme rates were usually determined at much lower substrate concentrations.

The rate of formation of US by the extracts was found to be fairly con- stant over a period of 2 hours with a slight decrease in rate beyond that period. The enzyme was completely inactivated by heating for 2 minutes

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754 PYRIMIDINE BIOSYNTHESIS IN E. COLI

at loo”, and addition of inactivated extract to the reaction mixtures had no stimulatory effect. Magnesium ion did not activate the reaction, which proceeded at an optimal rate even in 0.2 M NaF, but instead became in- hibitory above concentrations of 0.01 M.

When the rate of US synthesis was studied as a function of aspartate and of carbamyl phosphate concentration, the results shown in Fig. 6 were obtained. K, of carbamyl phosphate was calculated to be 4.3 X 10V3 M, and K, of aspartic acid was 2.5 X 1O-2 M (Fig. 6) at concentrations of 0.005 M L-aspartate and of 0.01 M carbamyl phosphate, respectively. From the same plots, the maximal velocities of US synthesis can be obtained, and the maximal velocity of US synthesis at both saturating carbamyl phosphate and saturating aspartic acid concentrations can be calculated to be 9.1 X lo-l6 mole per cell per hour. This value is far larger than that of 6.7 X 10-l’ mole per cell per hour necessary to allow for the observed growth rate of the cells, and need for the high enzyme level may be ex- plained, in part, by the high K, for aspartic acid. The actual concentra- tion of aspartic acid in the cell probably is much lower than that required to saturate the enzyme.

DISCUSSION

The work reported here supports and amplifies the scheme shown at the start of this paper. The two enzymes catalyzing the reversible con- versions US $ DHO C+ OA were first reported by Lieberman and Korn- berg to be present in an organism, 2. oroticum, grown with OA as a carbon source. As shown here, however, 2. oroticum cultured in glucose medium grew faster than it did when OA was used, yet contained only small amounts of the two enzymes. This result indicated the adaptive formation of the enzymes to metabolize OA for general metabolic purposes and raised the question of whether these enzymes were involved in pyrimidine bio- synthesis. The studies with E. coli in vitro, however, showed activities of ureidosuccinic acid synthet,ase, dihydroorotase, and dihydroorotic acid dehydrogenase more than enough to account for the growth rates of the whole cells. The low activities of the enzymes in 2. oroticum grown in glucose medium were probably adequate for the low growth rate of the cells, however, although quantitative study was difficult.

Since one substrain of E. coli mutant 6386, which lacks the enzyme di- hydroorotic acid dehydrogenase, was able to grow on OA at the normal rate for wild type cells, attachment of ribose (9-11) at a step prior to OA formation was not necessary to account for pyrimidine formation. Sum- mation of the work in vivo and in vitro shows that the scheme at the start of this paper is valid for E. coli and can explain the total observed growth rate of the cells.

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R. A. YATES AND A. B. PARDEE 755

SUMMARY

1. One pyrimidine-requiring Escherichia coli mutant has been charac- terized as lacking the enzyme dihydroorotic acid dehydrogenase. This mutant does not produce erotic acid, but can use it as a growth factor. Another mutant uses uracil and cytosine as growth factors and excretes ureidosuccinic acid (US), dihydroorotic acid (DHO), and erotic acid (OA) in the absence of pyrimidine growth factors.

2. The rates of production of total pyrimidine intermediates by the two mutants in the absence of uracil or cytosine were nearly identical and were slightly greater than the rate of utilization of pyrimidine for optimal nucleic acid formation and growth of the culture.

3. Cell-free extracts of Zymobacterium oroticum grown in glucose medium had about one-twentieth the dihydroorotase and dihydroorotic dehydro- genase activities of extracts of cells grown in OA medium.

4. Cell-free extracts of E. co& strain B, grown in minimal media con- verted aspartic acid plus carbamyl phosphate to US, US to DHO, and DHO to OA several times faster than necessary to account for observed pyrimi- dine biosynthesis in growing cells. This evidence, plus the results in vivo, indicates that these conversions are part of the pathway for pyrimidine biosynthesis in E. coli.

5. pH optima, substrate affinities, and maximal rates of reaction were determined for the above conversions.

6. A convenient assay for DHO is reported.

BIBLIOGRAPHY

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756 PYRIMIDINE BIOSYNTHESIS IN E. COLI

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Richard A. Yates and Arthur B. PardeeESCHERICHIA COLI

PYRIMIDINE BIOSYNTHESIS IN

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