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[7] Aspartate carbamyltransferase (Pseudomonas fluorescens)
[7]
ASPARTATE CARBAMYLTRANSFERASE
[7]
Aspartate
Carbamyltransferase fluorescens)
51
(Pseudornonas
B y L I N D A B . A D A I R a n d MARY E L L E N JONES Aspartate + carbamyl phosphate v-~carbamyl aspartate + P~ - H +
In preliminary studies 1 aspartate carbamyltransferase from Pseudomonas fluorescens exhibited kinetic and feedback inhibition characteristics different from the ATCase of other bacteria. The enzyme from this bacteria was thus isolated and its characteristics studied. 2 The procedures to be described are derived from an earlier fractionation procedure. 2 Assay Methods
Method I A colorimetric method of the determination of ATCase 3 was used for following the purification. For this method the enzyme assay solution contained (unless otherwise stated) 100 mM Tris-C1, pH 8.5; 10 mM aspartate, pH 8.5; 1 mM lithium carbamyl phosphate; and enzyme in a final volume of 1 ml. The reaction mixture, minus enzyme, was incubated for 1 min. The enzyme was added, and the reaction incubated for 10 min. The reaction was stopped by the addition of 1 ml of color mixture. The color mixture was prepared immediately before use and consisted of 2 parts of the antipyrine/H~SO4 reagent and 1 part of the oxime reagent below. 1. Antipyrine/H2SO4 reagent: 5 g/liter of antipyrine in 50% (v/v) sulfuric acid. 2. Oxime reagent: 0.80 g of diacetylmonoxime in 100 ml of 5% (v/v) acetic acid. This reagent should be stored at 4 ° in a dark bottle covered in aluminum foil. After the color mixture was added to the enzyme reaction, the tubes were capped with marbles and placed in a 60 ° water bath for 120 min. The absorbancy at 466 nm was determined and compared with the color produced by standard amounts of carbamyl L-aspartate. 1 j. Neumann and M. E. Jones, Arch. Biochem. Biophys. 104, 438-477 (1964). 2 L. B. Adair and M. E. Jones, J. Biol. Chem. 247, 2308-2315 (1972). 3 L. M. Prescott and M. E. Jones, Anal. Biochem. 32, 408-419 (1969). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5
52
De Novo PYRIMIDINE BIOSYNTHESIS
[7]
Method H
When low concentrations (below 20/zM) of CAP carbamyl phosphate were used in the kinetic studies, the radioactive assay of Bethell et al. 4 must be used. The [14C]CAP used for this assay was obtained from New England Nuclear (1.97 mCi/mmole). Before use it was recrystallized from cold ethanol since 10% of the total radioactivity was a contaminant. Nine volumes of ice-cold absolute ethanol were added to a 0.1 to 0.2 M [14C]CAP solution. The material remained at 0 ° for 15 min. The precipitated [14C]CAP was removed by vacuum filtration, washed once with cold 95% ethanol and washed once with cold ether. The CAP was dried rapidly by placing in a 25 ° vacuum desiccator with P205. After 30 niin, the P205 was stirred to give a new surface. This procedure was repeated until ether vapors could not be detected and the P205 remained dry. The CAP was dissolved in cold water to yield a 0.1 to 0.2 M solution. The solution was divided into 10 bd aliquots that were frozen in separate tubes. The reaction mixture contained 100 mM Tris-Cl, pH 8.5, 10 mM aspartate, pH 8.5, and the desired concentration of [~4C]CAP in a final volume of 1 ml. The mixture, minus enzyme, was preincubated for 1 rain at 30°, the enzyme added, and the reaction allowed to proceed for 10 min. The reaction was stopped by the addition of 0.1 ml of 70% perchloric acid, and the tube was capped and heated for 3 min in a 100° water bath. After cooling the tube, COs gas was bubbled through the solution for 30 min. The entire sample was transferred to a scintillation vial containing a napthalene-dioxane mixture and counted. Protein concentration was determined by the method of Oyama and Eagle.5 A unit of enzyme activity is defined as that amount of enzyme that catalyzes the formation of 1 /zmole of carbamyl aspartate per minute. The specific activity of the enzyme is the number of units of activity per mg of protein. The activity of the enzyme varies greatly with pH. The optimum activity of the enzyme is observed at p H 8.52. Purification All purification steps were carried out at 4° and at no time after the frozen bacterial cells were initially disrupted was the enzyme frozen. Step 1. Crude Extract. Seventy-five grams of frozen cell paste of Pseudomonas fluorescens (General Biochemicals, cat no. 150300) were 4 M. R. Bethe|l, K. E. Smith, J. S. White, and M. E. Jones, Proc. Natl. Acad. Sci. U.S.A. 60, 1442-1449 (1968). 5 V. I. Oyama and H. Eagle, Proc. Soc. Exp. Biol. Med. 91, 305-307 (1956).
[7]
ASPARTATECARBAMYLTRANSFERASE
53
thawed and suspended in 50 ml of homogenizing buffer (10 mM potassium phosphate, pH 7.5; 0.5 mM mercaptoethanol; and 20 txM EDTA). The cells were disrupted by passing the suspended cells twice through a French press under a pressure of 16,000 psi. The resulting homogenate was then centrifuged for 1 hr at 15,000 g.
Step 2. Streptomycin Sulfate Addition. To the supernatant was slowly added with vigorous stirring 50 ml of a solution containing 10% streptomycin sulfate and 10 mM potassium phosphate, pH 7.5. The resulting precipitate was allowed to settle for 1 hr and then removed by centrifuging for 30 rain at 15,000 g. The supernatant was dialyzed overnight against two changes of 4 liters each of homogenizing buffer. If further precipitation occurred, it was removed by centrifugation. Step 3. Ammonium Sulfate Fractionation. To dialyzed enzyme from step 2, 24.6 g of solid ammonium sulfate were added slowly, resulting in 30% saturation. After settling for 1 hr the precipitate was removed by centrifugation and the supernatant was brought to 43% saturation by the further addition of 11.2 g ammonium sulfate. The suspension was allowed to settle for 3 hr, and the precipitate containing the enzyme was collected by centrifugation. The pellet was dissolved in 6 ml of homogenizing buffer and dialyzed overnight against two changes of the homogenizing buffer. Step 4. Sephadex G-200 Chromatography. About 4 ml of the dialyzed enzyme from step 3 containing 40 mg protein per ml were applied to the bottom of a Pharmacia column, 100 x 2.5 cm containing Sephadex G2013. The Sephadex G-200 slurry was prepared 6 by adding 25 g of Sephadex G-200 slowly into a 2-liter beaker containing 1.3 liters of rapidly boiling buffer (10 mM potassium phosphate, pH 7.5, and 50 mM KCI). The heat source was removed after half of the Sephadex had been added, and stirring was continued throughout addition until no lumps were visible. The suspension was cooled, the fines decanted, and the resulting slurry used to pack the column. After the enzyme was applied, the column was regulated to a flow rate of 18 ml/hr and fractions of 3.5 ml were collected and concentrated in an Amicon ultrafiltration cell fitted with a PM-10 membrane to a concentration of about 2.5 mg of protein per ml. At this point the enzyme was stored until several batches of enzyme could be brought to this stage of purity. The enzyme is very stable in this condition. Step 5. Hydroxylapatite Chromatography. Hydroxylapatite was obtained commercially or prepared using a modification of the methods of 6 M. R. Bethell and M. E. J o n e s , Arch. Biochem. Biophys. 134, 352-365 (1969).
54
De Novo PYRIMIDINEBIOSYNTHESIS
[7]
Siegelman et al. r and Sizer and Jenkins, s as described by Prescott. 9 The hydroxylapatite for column use was prepared simply by suspending it in 5 mM potassium phosphate buffer at p H 7.5 and pouring directly into a column. The column with a fritted glass lower support was 8 cm in diameter and was fitted with Whatman No. 1 filter paper. The hydroxylapatite was poured to a height of 2.5 cm and filter paper placed on top of that to prevent disturbing the bed. If the column was poured to a greater height, the flow rate was extremely slow. With a large-diameter column, a large quantity of the enzyme could be purified at one time. The enzyme from step 4 was dialyzed overnight against a 5 mM potassium phosphate buffer, pH 7.5. About 40 ml (2.5 mg protein per rrd) of the enzyme were layered on the column. The colunm was then washed with 700 ml of 5 mM phosphate buffer. Then 15 mM potassium phosphate buffer, p H 7.5, were then added and 10 ml fractions collected. The column maintained a flow rate of 60 ml/hr under a constant pressure of 5 cm buffer. Fractions with ATCase activity were collected and concentrated in an Amicon filtration cell to a protein concentration of 1 mg/ml. The column could be generated again by washing with 4 liters of 0.4 M potassium phosphate and then re-equilibrating with 5 mM potassium phosphate. With a regenerated column, the ATCase came off in the 5 mM phosphate wash, but appeared to have the same purity (as judged by electrophoresis) and specific activity as that coming off in the 15 mM phosphate wash with a freshly prepared column. Step 6. Preparative Polyacrylamide Gel Electrophoresis. The method used for the preparative gel electrophoresis was that described in the instruction manual of the "Poly-Prep" electrophoresis apparatus by Buchler Instruments, Inc. The procedures are fully described in the Trisglycine system of Jovin, Chrambach, and Naughton. 10 The height of the resulting gel was 1. I cm. This was the shortest gel that would adhere to the column support. The enzyme from step 5 (4 mg) was diluted to 20 ml in 50 mM Tris buffer, pH 8.9. To this were added 0.6 g of sucrose and 0.1 ml of 0.01% (v/v) Bromphenol blue. Using a current of 50 mA, the enzyme was eluted from the gel 17.5 hr after elution of the Bromphenol blue. The 7 H. W. Siegelman, G. A. Weiczorek, and B. C. Turner, Anal. Biochem. 13, 402-404
(1965). s I. W. Sizer, and W. T. Jenkins, in "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.), Vol. 5, pp. 677-684. Academic Press, New York, 1962. L. M. Prescott, Ph.D. Dissertation, Brandeis University, Waltham, Massachusetts (1%9). 10T. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351-369 (1964).
[7]
ASPARTATE CARBAMYLTRANSFERASE
55
tubes with enzyme activity were combined and again concentrated in an Amicon ultrafiltration cell. The purity of the ATCase was determined by two criteria: sedimentation equilibrium studies and analytical disc gel electrophoresis. Sedimentation to equilibrium performed according to the method of Yphantis lz and described in Adair and Jones ~ showed only one protein species to be present. Analytical disc gel electrophoresis carried out by the procedure of Davis la showed one protein band, and this band coincided directly with enzymic activity. Physical Properties M o l e c u l a r Weight
The molecular weight was determined by sedimentation to equilibrium 12 and by sucrose gradient centrifugation. 14 The sedimentation to equilibrium showed the enzyme to have a molecular weight of 360,000. The molecular weight by sucrose gradient centrifugation was found to be 365,000. Subunit Structure
When the enzyme from step 6 was reduced with sodium dodecyl sulfate (SDS) by the method of Weber and Osborn 1~ and then subjected to electrophoresis, a single band was obtained with a molecular weight of 180,000. The experiment was repeated after incubating the enzyme in 1% SDS and 1% mercaptoethanol for 4 hr followed by a 12-hr dialysis in 0. I% SDS and 0.1% mercaptoethanol; the same subunit weight was obtained. Renaturation o f ATCase
The enzyme (fraction 5) denatured with SDS as described above had no ATCase activity. 2 The activity was measured by the radioactive assay since SDS interferes with the color development of the normal assay. The denatured enzyme was then dialyzed against 5 liters of 10 mM sodium phosphate, pH 7.0, at room temperature. The buffer was changed every hour for 4 hr. The enzyme was then dialyzed against 5 liters of cold buffer for 36 hr. After the dialysis 1% of the activity ~ S. E. Hager and M. E. Jones, J. Biol. Chem. 240, 5667-5673 (1%7). See Table I, this
chapter. ~zD. A. Yphantis, Biochemistry 3, 297-317(1964). 13B. J. Davis, Ann. N. Y. Acad. Sci. 121, 404-427(1964). ~4R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372-1379(1%1). 15K. Weber and M. Osborn, J. Biol. Chem. 244, 4406-4412(1969).
56
De N o v o PYRIM1DINE BIOSYNTHESIS
[7]
¢-q
% z "
0
0
C)
o= C ~ ~
c~
~L ~
< <
z b<
C
.o
as
"--
0
0
~
C~
~-~ 0
~ . _ ~
=~<~ -=E>>~
.-
t.
~
"~
"--
[7]
ASPARTATE CARBAMYLTRANSFERASE
57
returned, and sucrose gradient centrifugation showed the enzyme to have a molecular weight of 360,000.
Carbamyl-phosphate Synthetase Activity Yeast xa and Neurospora, 17 are known to possess an ATCase-carbamyl-phosphate synthetase complex. To see if P. fluorescens had a similar complex, CPSase activity was measured at each stage of the purification process. Since the CPSase activity declines (Table I) at each step and cannot be detected in the purified enzyme, it is assumed that no such complex exists in the pseudomonads.
Kinetics Substrate saturation curves of ATCase from some strains of the pseudomonads show hyperbolic curves with limiting CAP, but become T A B L E II NUCLEOTIDE INHIBITION CONSTANTSa
Inhibitor CTP UTP ATP GTP CDP PP~ Pi Ribose-5-P CMP Cytosine Cytidine
K~b when CAP is limiting 0zM)
Ki c when aspartate is limiting (mM)
7.0 8.5 8.5 a 100 0.4 100 1500 N o inhibition s N o inhibition s N o inhibition s
No No No No No No
7.5 9.0 9.0 7.0 6.0 inhibition e inhibition s inhibition s inhibition s inhibition s inhibition s
a From Adair and Jones. 2 b Competitive inhibition with CAP limiting, i.e., 7.5, 15, and 30 p.M with aspartate = 10 raM. c Noncompetitive inhibition with aspartate limiting, i.e., i, 2, or 4 mM with CAP = 10 mM. a Ki for GTP could not be determined because all sources of GTP tested had PP~ as a contaminant. Since the Ki for PPi is so low the results with GTP were invalid. e ppi does not inhibition below 5 m M PP~ when the CAP concentration is 10 raM. Above 5 m M PPi, it begins to compete with CAP. s Tested as high as 10 m M with no inhibition. 16 p. F. Lue and J. G. Kaplan, Can. J. Biochern. 48, 155-159(1970). 1~ L. G. Williams, S. Bernhardt, and R. H. Davis, Biochemistry 9, 432%4335 (1970).
58
De Novo
PYRIMIDINEBIOSYNTHESIS
[8]
sigmoidal in the presence of the inhibitor UTP.1 The enzyme from the bacteria supplied by General Biochemicals is also inhibited by UTP and other nucleotides, but the saturation curve for C A P in the presence of UTP is hyperbolic and not sigmoidal. From Lineweaver-Burk double reciprocal plots, the Km for CAP was 14 pdk/ and the apparent Km for aspartate was 2.75 mM 2. Inhibitor studies done when either CAP or aspartate were limiting gave the results seen in Table II. When CAP was the limiting substrate the enzyme showed a similar inhibition with CTP, UTP, and ATP. However, PP~ was a powerful inhibitor. From this and other data ~ it appears the PP~ is the most important factor in the inhibition with limiting CAP, and the structure of the base itself is relatively unimportant. The inhibition of the enzyme by the nucleotides with limiting CAP is competitive. 2 By contrast, with limiting aspartate and saturating CAP, the nucleotides inhibit noncompetitively. Again, all the nucleotides inhibit to the same extent, but PP~, which inhibits strongly with limiting CAP, no longer inhibits. Acknowledgment This work was supported by National Science Foundation Grant GB7929, and by Grants HD-02148 and 5 F02HIM0300from the NationalInstitutesof Health.
[8] D i h y d r o o r o t a t e
Dehydrogenase
(Escherichia
coli) 1
By DORIS KARIBIAN
O
O
O~.~COO. ~- O L N ~ ] COO. Dihydroorotate ---,orotate + 2 H+ Assay Methods The enzyme is membrane-bound and linked with the electron transport system of the cell s. When the system is intact the enzyme can 1R. A. Yates and A. B. Pardee, Biochim. Biophys. Acta 221, 743-756 (1956). 2 W. H. Taylorand M. L. Taylor,J. Bacteriol. 88, 105-110 (1964). M E T H O D S I N E N Z Y M O L O G Y , VOL. L I
Copyright© 1978by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181951-5
ASPARTATE CARBAMYLTRANSFERASE
[7]
Aspartate
Carbamyltransferase fluorescens)
51
(Pseudornonas
B y L I N D A B . A D A I R a n d MARY E L L E N JONES Aspartate + carbamyl phosphate v-~carbamyl aspartate + P~ - H +
In preliminary studies 1 aspartate carbamyltransferase from Pseudomonas fluorescens exhibited kinetic and feedback inhibition characteristics different from the ATCase of other bacteria. The enzyme from this bacteria was thus isolated and its characteristics studied. 2 The procedures to be described are derived from an earlier fractionation procedure. 2 Assay Methods
Method I A colorimetric method of the determination of ATCase 3 was used for following the purification. For this method the enzyme assay solution contained (unless otherwise stated) 100 mM Tris-C1, pH 8.5; 10 mM aspartate, pH 8.5; 1 mM lithium carbamyl phosphate; and enzyme in a final volume of 1 ml. The reaction mixture, minus enzyme, was incubated for 1 min. The enzyme was added, and the reaction incubated for 10 min. The reaction was stopped by the addition of 1 ml of color mixture. The color mixture was prepared immediately before use and consisted of 2 parts of the antipyrine/H~SO4 reagent and 1 part of the oxime reagent below. 1. Antipyrine/H2SO4 reagent: 5 g/liter of antipyrine in 50% (v/v) sulfuric acid. 2. Oxime reagent: 0.80 g of diacetylmonoxime in 100 ml of 5% (v/v) acetic acid. This reagent should be stored at 4 ° in a dark bottle covered in aluminum foil. After the color mixture was added to the enzyme reaction, the tubes were capped with marbles and placed in a 60 ° water bath for 120 min. The absorbancy at 466 nm was determined and compared with the color produced by standard amounts of carbamyl L-aspartate. 1 j. Neumann and M. E. Jones, Arch. Biochem. Biophys. 104, 438-477 (1964). 2 L. B. Adair and M. E. Jones, J. Biol. Chem. 247, 2308-2315 (1972). 3 L. M. Prescott and M. E. Jones, Anal. Biochem. 32, 408-419 (1969). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5
52
De Novo PYRIMIDINE BIOSYNTHESIS
[7]
Method H
When low concentrations (below 20/zM) of CAP carbamyl phosphate were used in the kinetic studies, the radioactive assay of Bethell et al. 4 must be used. The [14C]CAP used for this assay was obtained from New England Nuclear (1.97 mCi/mmole). Before use it was recrystallized from cold ethanol since 10% of the total radioactivity was a contaminant. Nine volumes of ice-cold absolute ethanol were added to a 0.1 to 0.2 M [14C]CAP solution. The material remained at 0 ° for 15 min. The precipitated [14C]CAP was removed by vacuum filtration, washed once with cold 95% ethanol and washed once with cold ether. The CAP was dried rapidly by placing in a 25 ° vacuum desiccator with P205. After 30 niin, the P205 was stirred to give a new surface. This procedure was repeated until ether vapors could not be detected and the P205 remained dry. The CAP was dissolved in cold water to yield a 0.1 to 0.2 M solution. The solution was divided into 10 bd aliquots that were frozen in separate tubes. The reaction mixture contained 100 mM Tris-Cl, pH 8.5, 10 mM aspartate, pH 8.5, and the desired concentration of [~4C]CAP in a final volume of 1 ml. The mixture, minus enzyme, was preincubated for 1 rain at 30°, the enzyme added, and the reaction allowed to proceed for 10 min. The reaction was stopped by the addition of 0.1 ml of 70% perchloric acid, and the tube was capped and heated for 3 min in a 100° water bath. After cooling the tube, COs gas was bubbled through the solution for 30 min. The entire sample was transferred to a scintillation vial containing a napthalene-dioxane mixture and counted. Protein concentration was determined by the method of Oyama and Eagle.5 A unit of enzyme activity is defined as that amount of enzyme that catalyzes the formation of 1 /zmole of carbamyl aspartate per minute. The specific activity of the enzyme is the number of units of activity per mg of protein. The activity of the enzyme varies greatly with pH. The optimum activity of the enzyme is observed at p H 8.52. Purification All purification steps were carried out at 4° and at no time after the frozen bacterial cells were initially disrupted was the enzyme frozen. Step 1. Crude Extract. Seventy-five grams of frozen cell paste of Pseudomonas fluorescens (General Biochemicals, cat no. 150300) were 4 M. R. Bethe|l, K. E. Smith, J. S. White, and M. E. Jones, Proc. Natl. Acad. Sci. U.S.A. 60, 1442-1449 (1968). 5 V. I. Oyama and H. Eagle, Proc. Soc. Exp. Biol. Med. 91, 305-307 (1956).
[7]
ASPARTATECARBAMYLTRANSFERASE
53
thawed and suspended in 50 ml of homogenizing buffer (10 mM potassium phosphate, pH 7.5; 0.5 mM mercaptoethanol; and 20 txM EDTA). The cells were disrupted by passing the suspended cells twice through a French press under a pressure of 16,000 psi. The resulting homogenate was then centrifuged for 1 hr at 15,000 g.
Step 2. Streptomycin Sulfate Addition. To the supernatant was slowly added with vigorous stirring 50 ml of a solution containing 10% streptomycin sulfate and 10 mM potassium phosphate, pH 7.5. The resulting precipitate was allowed to settle for 1 hr and then removed by centrifuging for 30 rain at 15,000 g. The supernatant was dialyzed overnight against two changes of 4 liters each of homogenizing buffer. If further precipitation occurred, it was removed by centrifugation. Step 3. Ammonium Sulfate Fractionation. To dialyzed enzyme from step 2, 24.6 g of solid ammonium sulfate were added slowly, resulting in 30% saturation. After settling for 1 hr the precipitate was removed by centrifugation and the supernatant was brought to 43% saturation by the further addition of 11.2 g ammonium sulfate. The suspension was allowed to settle for 3 hr, and the precipitate containing the enzyme was collected by centrifugation. The pellet was dissolved in 6 ml of homogenizing buffer and dialyzed overnight against two changes of the homogenizing buffer. Step 4. Sephadex G-200 Chromatography. About 4 ml of the dialyzed enzyme from step 3 containing 40 mg protein per ml were applied to the bottom of a Pharmacia column, 100 x 2.5 cm containing Sephadex G2013. The Sephadex G-200 slurry was prepared 6 by adding 25 g of Sephadex G-200 slowly into a 2-liter beaker containing 1.3 liters of rapidly boiling buffer (10 mM potassium phosphate, pH 7.5, and 50 mM KCI). The heat source was removed after half of the Sephadex had been added, and stirring was continued throughout addition until no lumps were visible. The suspension was cooled, the fines decanted, and the resulting slurry used to pack the column. After the enzyme was applied, the column was regulated to a flow rate of 18 ml/hr and fractions of 3.5 ml were collected and concentrated in an Amicon ultrafiltration cell fitted with a PM-10 membrane to a concentration of about 2.5 mg of protein per ml. At this point the enzyme was stored until several batches of enzyme could be brought to this stage of purity. The enzyme is very stable in this condition. Step 5. Hydroxylapatite Chromatography. Hydroxylapatite was obtained commercially or prepared using a modification of the methods of 6 M. R. Bethell and M. E. J o n e s , Arch. Biochem. Biophys. 134, 352-365 (1969).
54
De Novo PYRIMIDINEBIOSYNTHESIS
[7]
Siegelman et al. r and Sizer and Jenkins, s as described by Prescott. 9 The hydroxylapatite for column use was prepared simply by suspending it in 5 mM potassium phosphate buffer at p H 7.5 and pouring directly into a column. The column with a fritted glass lower support was 8 cm in diameter and was fitted with Whatman No. 1 filter paper. The hydroxylapatite was poured to a height of 2.5 cm and filter paper placed on top of that to prevent disturbing the bed. If the column was poured to a greater height, the flow rate was extremely slow. With a large-diameter column, a large quantity of the enzyme could be purified at one time. The enzyme from step 4 was dialyzed overnight against a 5 mM potassium phosphate buffer, pH 7.5. About 40 ml (2.5 mg protein per rrd) of the enzyme were layered on the column. The colunm was then washed with 700 ml of 5 mM phosphate buffer. Then 15 mM potassium phosphate buffer, p H 7.5, were then added and 10 ml fractions collected. The column maintained a flow rate of 60 ml/hr under a constant pressure of 5 cm buffer. Fractions with ATCase activity were collected and concentrated in an Amicon filtration cell to a protein concentration of 1 mg/ml. The column could be generated again by washing with 4 liters of 0.4 M potassium phosphate and then re-equilibrating with 5 mM potassium phosphate. With a regenerated column, the ATCase came off in the 5 mM phosphate wash, but appeared to have the same purity (as judged by electrophoresis) and specific activity as that coming off in the 15 mM phosphate wash with a freshly prepared column. Step 6. Preparative Polyacrylamide Gel Electrophoresis. The method used for the preparative gel electrophoresis was that described in the instruction manual of the "Poly-Prep" electrophoresis apparatus by Buchler Instruments, Inc. The procedures are fully described in the Trisglycine system of Jovin, Chrambach, and Naughton. 10 The height of the resulting gel was 1. I cm. This was the shortest gel that would adhere to the column support. The enzyme from step 5 (4 mg) was diluted to 20 ml in 50 mM Tris buffer, pH 8.9. To this were added 0.6 g of sucrose and 0.1 ml of 0.01% (v/v) Bromphenol blue. Using a current of 50 mA, the enzyme was eluted from the gel 17.5 hr after elution of the Bromphenol blue. The 7 H. W. Siegelman, G. A. Weiczorek, and B. C. Turner, Anal. Biochem. 13, 402-404
(1965). s I. W. Sizer, and W. T. Jenkins, in "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.), Vol. 5, pp. 677-684. Academic Press, New York, 1962. L. M. Prescott, Ph.D. Dissertation, Brandeis University, Waltham, Massachusetts (1%9). 10T. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351-369 (1964).
[7]
ASPARTATE CARBAMYLTRANSFERASE
55
tubes with enzyme activity were combined and again concentrated in an Amicon ultrafiltration cell. The purity of the ATCase was determined by two criteria: sedimentation equilibrium studies and analytical disc gel electrophoresis. Sedimentation to equilibrium performed according to the method of Yphantis lz and described in Adair and Jones ~ showed only one protein species to be present. Analytical disc gel electrophoresis carried out by the procedure of Davis la showed one protein band, and this band coincided directly with enzymic activity. Physical Properties M o l e c u l a r Weight
The molecular weight was determined by sedimentation to equilibrium 12 and by sucrose gradient centrifugation. 14 The sedimentation to equilibrium showed the enzyme to have a molecular weight of 360,000. The molecular weight by sucrose gradient centrifugation was found to be 365,000. Subunit Structure
When the enzyme from step 6 was reduced with sodium dodecyl sulfate (SDS) by the method of Weber and Osborn 1~ and then subjected to electrophoresis, a single band was obtained with a molecular weight of 180,000. The experiment was repeated after incubating the enzyme in 1% SDS and 1% mercaptoethanol for 4 hr followed by a 12-hr dialysis in 0. I% SDS and 0.1% mercaptoethanol; the same subunit weight was obtained. Renaturation o f ATCase
The enzyme (fraction 5) denatured with SDS as described above had no ATCase activity. 2 The activity was measured by the radioactive assay since SDS interferes with the color development of the normal assay. The denatured enzyme was then dialyzed against 5 liters of 10 mM sodium phosphate, pH 7.0, at room temperature. The buffer was changed every hour for 4 hr. The enzyme was then dialyzed against 5 liters of cold buffer for 36 hr. After the dialysis 1% of the activity ~ S. E. Hager and M. E. Jones, J. Biol. Chem. 240, 5667-5673 (1%7). See Table I, this
chapter. ~zD. A. Yphantis, Biochemistry 3, 297-317(1964). 13B. J. Davis, Ann. N. Y. Acad. Sci. 121, 404-427(1964). ~4R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372-1379(1%1). 15K. Weber and M. Osborn, J. Biol. Chem. 244, 4406-4412(1969).
56
De N o v o PYRIM1DINE BIOSYNTHESIS
[7]
¢-q
% z "
0
0
C)
o= C ~ ~
c~
~L ~
< <
z b<
C
.o
as
"--
0
0
~
C~
~-~ 0
~ . _ ~
=~<~ -=E>>~
.-
t.
~
"~
"--
[7]
ASPARTATE CARBAMYLTRANSFERASE
57
returned, and sucrose gradient centrifugation showed the enzyme to have a molecular weight of 360,000.
Carbamyl-phosphate Synthetase Activity Yeast xa and Neurospora, 17 are known to possess an ATCase-carbamyl-phosphate synthetase complex. To see if P. fluorescens had a similar complex, CPSase activity was measured at each stage of the purification process. Since the CPSase activity declines (Table I) at each step and cannot be detected in the purified enzyme, it is assumed that no such complex exists in the pseudomonads.
Kinetics Substrate saturation curves of ATCase from some strains of the pseudomonads show hyperbolic curves with limiting CAP, but become T A B L E II NUCLEOTIDE INHIBITION CONSTANTSa
Inhibitor CTP UTP ATP GTP CDP PP~ Pi Ribose-5-P CMP Cytosine Cytidine
K~b when CAP is limiting 0zM)
Ki c when aspartate is limiting (mM)
7.0 8.5 8.5 a 100 0.4 100 1500 N o inhibition s N o inhibition s N o inhibition s
No No No No No No
7.5 9.0 9.0 7.0 6.0 inhibition e inhibition s inhibition s inhibition s inhibition s inhibition s
a From Adair and Jones. 2 b Competitive inhibition with CAP limiting, i.e., 7.5, 15, and 30 p.M with aspartate = 10 raM. c Noncompetitive inhibition with aspartate limiting, i.e., i, 2, or 4 mM with CAP = 10 mM. a Ki for GTP could not be determined because all sources of GTP tested had PP~ as a contaminant. Since the Ki for PPi is so low the results with GTP were invalid. e ppi does not inhibition below 5 m M PP~ when the CAP concentration is 10 raM. Above 5 m M PPi, it begins to compete with CAP. s Tested as high as 10 m M with no inhibition. 16 p. F. Lue and J. G. Kaplan, Can. J. Biochern. 48, 155-159(1970). 1~ L. G. Williams, S. Bernhardt, and R. H. Davis, Biochemistry 9, 432%4335 (1970).
58
De Novo
PYRIMIDINEBIOSYNTHESIS
[8]
sigmoidal in the presence of the inhibitor UTP.1 The enzyme from the bacteria supplied by General Biochemicals is also inhibited by UTP and other nucleotides, but the saturation curve for C A P in the presence of UTP is hyperbolic and not sigmoidal. From Lineweaver-Burk double reciprocal plots, the Km for CAP was 14 pdk/ and the apparent Km for aspartate was 2.75 mM 2. Inhibitor studies done when either CAP or aspartate were limiting gave the results seen in Table II. When CAP was the limiting substrate the enzyme showed a similar inhibition with CTP, UTP, and ATP. However, PP~ was a powerful inhibitor. From this and other data ~ it appears the PP~ is the most important factor in the inhibition with limiting CAP, and the structure of the base itself is relatively unimportant. The inhibition of the enzyme by the nucleotides with limiting CAP is competitive. 2 By contrast, with limiting aspartate and saturating CAP, the nucleotides inhibit noncompetitively. Again, all the nucleotides inhibit to the same extent, but PP~, which inhibits strongly with limiting CAP, no longer inhibits. Acknowledgment This work was supported by National Science Foundation Grant GB7929, and by Grants HD-02148 and 5 F02HIM0300from the NationalInstitutesof Health.
[8] D i h y d r o o r o t a t e
Dehydrogenase
(Escherichia
coli) 1
By DORIS KARIBIAN
O
O
O~.~COO. ~- O L N ~ ] COO. Dihydroorotate ---,orotate + 2 H+ Assay Methods The enzyme is membrane-bound and linked with the electron transport system of the cell s. When the system is intact the enzyme can 1R. A. Yates and A. B. Pardee, Biochim. Biophys. Acta 221, 743-756 (1956). 2 W. H. Taylorand M. L. Taylor,J. Bacteriol. 88, 105-110 (1964). M E T H O D S I N E N Z Y M O L O G Y , VOL. L I
Copyright© 1978by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181951-5