- Email: [email protected]
[8] Dihydroorotate dehdydrogenase (Escherichia coli)
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
[8]
DIHYDROOROTATE DEHYDROGENASE
59
therefore be assayed either as an oxidase 1'3 or as a dehydrogenase. When the system is not intact the enzyme can be assayed as a dehydrogenase using such electron acceptors as ferricyanide, various quinones, or 2,6-dichlorophenolindophenol (DCIP). 2"4 The oxidase and DCIP-reducing methods are described here. They have both been used with extracts from B and K12 strains grown on various minimal salts and rich media. With particulate preparations the specific oxidase activity at pH 8.3 is usually about 40% higher than the specific DCIP-reducing activity at pH 7.0. If both assay methods are used in parallel it is convenient to use the same buffer, 100 mM Tris.HCl, pH 7.6, instead of the different ones indicated below. Anaerobically grown cells have very low activity.
Dihydroorotate Oxidase Principle. Enzyme activity is determined spectrophotometrically by following the rate of absorbance increase at 290 nm which is associated with the appearance of orotate. Reagents Tris'HC1, 1 M, pH 8.3, 0.3 ml Enzyme: crude extract or membrane fraction, 20 mg protein/ml, 150/.d Sodium dihydroorotate, 0.01 M, 0.3 ml; store cold
Procedure. To each of two 3-ml quartz cuvettes of 1-cm light path add, in order, buffer, distilled water, and enzyme. Let equilibrate at 25 ° in the spectrophotometer, adjust the absorbance of the control to zero at 290 nm, and then add substrate to the test cuvette with good mixing. Follow the rate of absorbance increase in the test cuvette. Specific activity is defined as nmoles of orotate formed per minute per milligram protein (assayed by the method of Lowry et al.5), taking the molar extinction of orotate at 290 nm as 6.2 × 103.1 Dihydroorotate Dehydrogenase Principle. Under conditions in which the bacterial oxidase system is inactive or absent, the enzyme transfers reducing groups from the substrate to the blue dye DCIP which on reduction no longer absorbs light at 600 nm. a j. R. Beckwith, A. B. Pardee, R. Austrian, and F. Jacob, J. Mol. Biol. 5, 618-634 (1962). 4 C. T. Kerr and R. W. Miller, J. Biol. Chem. 243, 2963-2968 (1968). 5 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265275 (1951).
60
De Novo PYRIMIDINE BIOSYNTHESIS
[8]
Reagents Sodium phosphate buffer, 1 M, pH 7.0, 0.3 ml KCN, 0.1 M, 0.15 ml Triton X-100, 1.0%, 0.3 ml; store cold DCIP, 0.001 M, 0.12 ml Sodium dihydroorotate, 0.01 M, 0.3 ml
Procedure. To three numbered 3-ml cuvettes of 1-cm light path add buffer, KCN, Triton X-100, water, and enzyme. Add DCIP to cuvettes 2 and 3, mix well, let equilibrate to 25 °, adjust absorbance of cuvette 1 to zero at 600 nm, add substrate to cuvettes 1 and 3, readjust control absorbance to zero, and follow rate of absorbance decrease in the DCIPcontaining cuvettes. Subtract the decrease observed in cuvette 2 from that in cuvette 3. With crude enzyme preparations the correction may be appreciable, especially during the first 2-3 min before stabilizing at an acceptably low level. With purified enzyme there is a negligible decrease in the absorbance of vessel 2. Use sufficient enzyme to give a net absorbance change of between 0.020 and 0.045/5 min during the first 10-15 min. Only initial rates are used. One unit of activity reduces 1 nmole DCIP per minute. Specific activity is expressed as units/mg protein taking the E600nm for DCIP as 20 x 103. Purification Procedure Although this procedure was developed with a p y r E - derivative of K12 strain AT12436 other derepressible o r leaky pyr- strains (except pyrD-) should do.
Growth Conditions. Cells are grown aerobically at 37 ° on medium containing 50 mM sodium-potassium phosphate (pH 7.0), 0.2% ammonium sulfate, 0.01% MgSO,'7 H20, 0.001% CaCI~, 0.2% Difco Casamino acids (technical), 0.2% glucose (autoclaved and added separately), and 8 mg uracil per liter. When the uracil is depleted growth stops (or is reduced), and culture is incubated 2 more hours with aeration before the cells are harvested. Derepression of the pyrimidine pathway increases cellular dihydroorotate dehydrogenase activity 5-10-fold. Freezing the cells does not impair activity. Unless otherwise stated all steps are carried out at 0-4 °. Membrane Particle Preparation. The cells (20-200 g wet weight) are washed in 40 mM Tris-HCl, pH 7.6, suspended in the same buffer (2g/ 8 D. Karibian and P. Couchoud, Biochim. Biophys. Acta 364, 218-232 (1974).
[8]
61
DIHYDROOROTATE DEHYDROGENASE
10ml), and broken in a French pressure cell at 4 tons/in 2. The extract is treated at room temperature with deoxyribonuclease and ribonuclease (1 txg each/ml) until the solution is no longer viscous, and it is then centrifuged at 100,000 g for 1 hr. The pellet is washed twice with onefifth the original volume of the Tris buffer, and it is then resuspended in 4 mM phosphate (pH 7.0)-5 mM MgClz to yield a protein concentration of 3 mg/ml.
Solubilization. Add 1 ml of 10% Triton X-100 per 100 ml and centrifuge at 150,000 g for 1 hr; discard the pellet. The supernatant is concentrated in a Diaflo filter with an XM50 membrane to a protein concentration of 5 mg/ml. Ammonium Sulfate Fractionation. Solid salt is added slowly with stirring to a concentration of 40% saturation. After 1 hr of stirring the preparation is centrifuged 30 min at 10,000 g. The supernatant is then brought to 50% saturation in the same way and recentrifuged. The pellet is resuspended in 10 mM Tris.HCl (pH 8.4)-0.01% Triton X-100 and dialyzed against the same buffer with ammonium sulfate added to 0.6 M. Since the enzyme is stable for months at this stage, and less so after the next two steps, 10-15 mg portions are taken for further purification. Agarose Filtration. The dialysate above is centrifuged at 150,000 g for 1 hr and the protein concentration adjusted to 3 mg/ml. The preparation is passed over a Biogel A-1.5, 200-400 mesh column (2.6 × 50 cm for a 3-5 ml sample) equilibrated with 10 mM Tris.HCl (pH 8.4)0.01% Triton X-100-0.6 M ammonium sulfate solution. Activity comes off at 1.8 void volumes. The best fractions are collected and concentrated on a Diaflo XM50 filter to 1-2 ml. PURIFICATIONPROCEDUREFOR DIHYDROOROTATEDEHYDROGENASE(E. coli)
Preparation Crude extract Washed particles Solubilized enzyme 150,000g supernatant Precipitate 40-50% saturation (NH4)2SO4 Agarose column peak DEAE-cellulose column
Total activity units (nmoles product per min)
Protein (mg)
141.5 83.5
18,900 1,326
102.0
246
38.2 18.1 6.8
37.4 6.6 1.8
Specific activity (units/rag) 7.5 63 414 1020 2730 3630
62
De Novo PYRIMIDINE BIOSYNTHESIS
[8]
DEAE-Cellulose Chromatography. The above concentrate is loaded on a DEAE-cellulose column (1.6 × 5 cm) equilibrated with 50 mM sodium-potassium phosphate (pH 6.8)-0.1 M ammonium sulfate and then washed with the column buffer. Fractions of 0.5 ml are taken in tubes containing 50 ~1 1% Triton X-100. Under these conditions the enzyme does not adhere to the column. Peak activity comes off at 2 column volumes. The specific activity attained varies from 400 to 800 times that of the crude extract. Since polyacrylamide gel electrophoresis shows very little contaminating protein in all cases, the variability is probably due to enzyme inactivation. Properties
Stability. All the above preparations through the ammonium sulfate step retain activity indefinitely at -18 °. The particulate enzyme is also heat stable at 60 ° when mM orotate or dihydroorotate is present. After solubilization of the enzyme Triton X-100 is indispensable. Stimulators and Inhibitors. Nonionic detergents such as Triton X100, Brij 35 (0.02%), and Nonidet P42 (0.4%), ammonium salts (0.4 M acetate, phosphate, and sulfate), and bovine serum albumin stimulate DCIP-reducing activity increasingly with purification. Triton X-100 and ammonium salts together stimulate synergistically. Phospholipids, especially diphosphatidylglycerol, stimulate solubilized activity.7 Deoxycholate, dodecyl sulfate, and free fatty acids inhibit. Orotate is a noncompetitive inhibitor in the absence, and a competitive inhibitor in the presence, of Triton X-100. pCMB treatment has no effect on activity. Molecular Weight. Molecular weight is estimated to be about 67,000 by agarose filtration.
Kinetics. Km(app) for dihydroorotate at pH 7.6 in the presence of Triton X-100 is 1 × 10-SM. Thermodynamics. Activation energies are 9.9 kcal and 16.8 kcal] mole, respectively, above and below the phase change temperature which is 19° in lipid-rich (> 0.15 ~moles lipid phosphate/mg protein) preparations and 15-16 ° in lipid-poor preparations with Triton X-100.
Other Properties. This enzyme does not contain flavins. There is evidence that the physiological electron acceptor is ubiquinone under 7 D. Karibian, Biochim. Biophys. Acta 302, 205-215 (1973).
[9]
DIHYDROOROTATE DEHYDROGENASE
63
aerobic conditions and menaquinone under anaerobic conditions, s The enzyme is associated with the inner side of the bacterial cytoplasmic membrane. It can be detached from it by the action of phospholipase As (Naja naja or pig pancreatic enzyme) followed by adjustment of the pH to 8.4. Enzyme solubilized in this way has, however, a strong tendency to form inactive aggregates. s N. A. Newton, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 244, 155-166 (1971).
[9] D i h y d r o o r o t a t e
Dehydrogenase
(Neurospora)
By R. W. MILLER Dihydroorotate + quinone ~ orotate + hydroquinone
Enzymes catalyzing the oxidation of dihydroorotate were originally identified in anaerobic bacteria 1 and mammalian cells 2 establishing the reaction as an obligatory step in the de novo synthesis of the pyrimidine heterocyclic ring. Constitutive enzymes responsible for catalyzing this step have been extensively investigated in prokaryotes 3 and fungi. 4 Although the molecular and catalytic properties of the enzyme differ depending on the source, biosynthetic dihydroorotate dehydrogenase (E.C. 1.3.3.15) is a mitochondrial enzyme in eukaryotic cells e'7 The oxidation of the substrate may be linked to reduction of components of the mitochondrial respiratory chain through quinones which are the primary electron acceptors for the enzyme purified as described in this article. The enzyme is a lipoprotein which can be isolated in catalytically active form from the mitochondrial membrane with nonionic detergents. Special treatment is required to prevent reaggregation or complete inactivation of the enzyme. Only dihydroorotate (2.5-4 mM) is capable of full protection of the isolated enzyme during purification. 6 Pyridine nucleotides cannot serve as electron acceptors for the fungal enzyme in situ nor can molecular oxygen serve as a primary electron acceptor for the isolated enzyme. 1 I. Lieberman and A. Kornberg, Biochim. Biophys. Acta 12, 223 (1953). 2 R. Wu and D. W. Wilson, J. Biol. Chem. 223, 195 (1956). 3 W. H. Taylor, M. L. Taylor, and D. F. Eames, J. Bacteriol. 91, 2251 (1966), 4 R. W. Miller, Arch. Biochem. Biophys. 146, 256 (1971). 5 See properties o f enzyme for consideration o f reactivity with oxygen. R. W. Miller, Can. J. Biochem. 53, 1288 (1975). r j. j. Chen and M. E. Jones, Arch. Biochem. Biophys. 176, 82 (1976).
M E T H O D S IN E N Z Y M O L O G Y , V O L . L I
Copyright ~) 1978by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181951
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
[8]
DIHYDROOROTATE DEHYDROGENASE
59
therefore be assayed either as an oxidase 1'3 or as a dehydrogenase. When the system is not intact the enzyme can be assayed as a dehydrogenase using such electron acceptors as ferricyanide, various quinones, or 2,6-dichlorophenolindophenol (DCIP). 2"4 The oxidase and DCIP-reducing methods are described here. They have both been used with extracts from B and K12 strains grown on various minimal salts and rich media. With particulate preparations the specific oxidase activity at pH 8.3 is usually about 40% higher than the specific DCIP-reducing activity at pH 7.0. If both assay methods are used in parallel it is convenient to use the same buffer, 100 mM Tris.HCl, pH 7.6, instead of the different ones indicated below. Anaerobically grown cells have very low activity.
Dihydroorotate Oxidase Principle. Enzyme activity is determined spectrophotometrically by following the rate of absorbance increase at 290 nm which is associated with the appearance of orotate. Reagents Tris'HC1, 1 M, pH 8.3, 0.3 ml Enzyme: crude extract or membrane fraction, 20 mg protein/ml, 150/.d Sodium dihydroorotate, 0.01 M, 0.3 ml; store cold
Procedure. To each of two 3-ml quartz cuvettes of 1-cm light path add, in order, buffer, distilled water, and enzyme. Let equilibrate at 25 ° in the spectrophotometer, adjust the absorbance of the control to zero at 290 nm, and then add substrate to the test cuvette with good mixing. Follow the rate of absorbance increase in the test cuvette. Specific activity is defined as nmoles of orotate formed per minute per milligram protein (assayed by the method of Lowry et al.5), taking the molar extinction of orotate at 290 nm as 6.2 × 103.1 Dihydroorotate Dehydrogenase Principle. Under conditions in which the bacterial oxidase system is inactive or absent, the enzyme transfers reducing groups from the substrate to the blue dye DCIP which on reduction no longer absorbs light at 600 nm. a j. R. Beckwith, A. B. Pardee, R. Austrian, and F. Jacob, J. Mol. Biol. 5, 618-634 (1962). 4 C. T. Kerr and R. W. Miller, J. Biol. Chem. 243, 2963-2968 (1968). 5 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265275 (1951).
60
De Novo PYRIMIDINE BIOSYNTHESIS
[8]
Reagents Sodium phosphate buffer, 1 M, pH 7.0, 0.3 ml KCN, 0.1 M, 0.15 ml Triton X-100, 1.0%, 0.3 ml; store cold DCIP, 0.001 M, 0.12 ml Sodium dihydroorotate, 0.01 M, 0.3 ml
Procedure. To three numbered 3-ml cuvettes of 1-cm light path add buffer, KCN, Triton X-100, water, and enzyme. Add DCIP to cuvettes 2 and 3, mix well, let equilibrate to 25 °, adjust absorbance of cuvette 1 to zero at 600 nm, add substrate to cuvettes 1 and 3, readjust control absorbance to zero, and follow rate of absorbance decrease in the DCIPcontaining cuvettes. Subtract the decrease observed in cuvette 2 from that in cuvette 3. With crude enzyme preparations the correction may be appreciable, especially during the first 2-3 min before stabilizing at an acceptably low level. With purified enzyme there is a negligible decrease in the absorbance of vessel 2. Use sufficient enzyme to give a net absorbance change of between 0.020 and 0.045/5 min during the first 10-15 min. Only initial rates are used. One unit of activity reduces 1 nmole DCIP per minute. Specific activity is expressed as units/mg protein taking the E600nm for DCIP as 20 x 103. Purification Procedure Although this procedure was developed with a p y r E - derivative of K12 strain AT12436 other derepressible o r leaky pyr- strains (except pyrD-) should do.
Growth Conditions. Cells are grown aerobically at 37 ° on medium containing 50 mM sodium-potassium phosphate (pH 7.0), 0.2% ammonium sulfate, 0.01% MgSO,'7 H20, 0.001% CaCI~, 0.2% Difco Casamino acids (technical), 0.2% glucose (autoclaved and added separately), and 8 mg uracil per liter. When the uracil is depleted growth stops (or is reduced), and culture is incubated 2 more hours with aeration before the cells are harvested. Derepression of the pyrimidine pathway increases cellular dihydroorotate dehydrogenase activity 5-10-fold. Freezing the cells does not impair activity. Unless otherwise stated all steps are carried out at 0-4 °. Membrane Particle Preparation. The cells (20-200 g wet weight) are washed in 40 mM Tris-HCl, pH 7.6, suspended in the same buffer (2g/ 8 D. Karibian and P. Couchoud, Biochim. Biophys. Acta 364, 218-232 (1974).
[8]
61
DIHYDROOROTATE DEHYDROGENASE
10ml), and broken in a French pressure cell at 4 tons/in 2. The extract is treated at room temperature with deoxyribonuclease and ribonuclease (1 txg each/ml) until the solution is no longer viscous, and it is then centrifuged at 100,000 g for 1 hr. The pellet is washed twice with onefifth the original volume of the Tris buffer, and it is then resuspended in 4 mM phosphate (pH 7.0)-5 mM MgClz to yield a protein concentration of 3 mg/ml.
Solubilization. Add 1 ml of 10% Triton X-100 per 100 ml and centrifuge at 150,000 g for 1 hr; discard the pellet. The supernatant is concentrated in a Diaflo filter with an XM50 membrane to a protein concentration of 5 mg/ml. Ammonium Sulfate Fractionation. Solid salt is added slowly with stirring to a concentration of 40% saturation. After 1 hr of stirring the preparation is centrifuged 30 min at 10,000 g. The supernatant is then brought to 50% saturation in the same way and recentrifuged. The pellet is resuspended in 10 mM Tris.HCl (pH 8.4)-0.01% Triton X-100 and dialyzed against the same buffer with ammonium sulfate added to 0.6 M. Since the enzyme is stable for months at this stage, and less so after the next two steps, 10-15 mg portions are taken for further purification. Agarose Filtration. The dialysate above is centrifuged at 150,000 g for 1 hr and the protein concentration adjusted to 3 mg/ml. The preparation is passed over a Biogel A-1.5, 200-400 mesh column (2.6 × 50 cm for a 3-5 ml sample) equilibrated with 10 mM Tris.HCl (pH 8.4)0.01% Triton X-100-0.6 M ammonium sulfate solution. Activity comes off at 1.8 void volumes. The best fractions are collected and concentrated on a Diaflo XM50 filter to 1-2 ml. PURIFICATIONPROCEDUREFOR DIHYDROOROTATEDEHYDROGENASE(E. coli)
Preparation Crude extract Washed particles Solubilized enzyme 150,000g supernatant Precipitate 40-50% saturation (NH4)2SO4 Agarose column peak DEAE-cellulose column
Total activity units (nmoles product per min)
Protein (mg)
141.5 83.5
18,900 1,326
102.0
246
38.2 18.1 6.8
37.4 6.6 1.8
Specific activity (units/rag) 7.5 63 414 1020 2730 3630
62
De Novo PYRIMIDINE BIOSYNTHESIS
[8]
DEAE-Cellulose Chromatography. The above concentrate is loaded on a DEAE-cellulose column (1.6 × 5 cm) equilibrated with 50 mM sodium-potassium phosphate (pH 6.8)-0.1 M ammonium sulfate and then washed with the column buffer. Fractions of 0.5 ml are taken in tubes containing 50 ~1 1% Triton X-100. Under these conditions the enzyme does not adhere to the column. Peak activity comes off at 2 column volumes. The specific activity attained varies from 400 to 800 times that of the crude extract. Since polyacrylamide gel electrophoresis shows very little contaminating protein in all cases, the variability is probably due to enzyme inactivation. Properties
Stability. All the above preparations through the ammonium sulfate step retain activity indefinitely at -18 °. The particulate enzyme is also heat stable at 60 ° when mM orotate or dihydroorotate is present. After solubilization of the enzyme Triton X-100 is indispensable. Stimulators and Inhibitors. Nonionic detergents such as Triton X100, Brij 35 (0.02%), and Nonidet P42 (0.4%), ammonium salts (0.4 M acetate, phosphate, and sulfate), and bovine serum albumin stimulate DCIP-reducing activity increasingly with purification. Triton X-100 and ammonium salts together stimulate synergistically. Phospholipids, especially diphosphatidylglycerol, stimulate solubilized activity.7 Deoxycholate, dodecyl sulfate, and free fatty acids inhibit. Orotate is a noncompetitive inhibitor in the absence, and a competitive inhibitor in the presence, of Triton X-100. pCMB treatment has no effect on activity. Molecular Weight. Molecular weight is estimated to be about 67,000 by agarose filtration.
Kinetics. Km(app) for dihydroorotate at pH 7.6 in the presence of Triton X-100 is 1 × 10-SM. Thermodynamics. Activation energies are 9.9 kcal and 16.8 kcal] mole, respectively, above and below the phase change temperature which is 19° in lipid-rich (> 0.15 ~moles lipid phosphate/mg protein) preparations and 15-16 ° in lipid-poor preparations with Triton X-100.
Other Properties. This enzyme does not contain flavins. There is evidence that the physiological electron acceptor is ubiquinone under 7 D. Karibian, Biochim. Biophys. Acta 302, 205-215 (1973).
[9]
DIHYDROOROTATE DEHYDROGENASE
63
aerobic conditions and menaquinone under anaerobic conditions, s The enzyme is associated with the inner side of the bacterial cytoplasmic membrane. It can be detached from it by the action of phospholipase As (Naja naja or pig pancreatic enzyme) followed by adjustment of the pH to 8.4. Enzyme solubilized in this way has, however, a strong tendency to form inactive aggregates. s N. A. Newton, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 244, 155-166 (1971).
[9] D i h y d r o o r o t a t e
Dehydrogenase
(Neurospora)
By R. W. MILLER Dihydroorotate + quinone ~ orotate + hydroquinone
Enzymes catalyzing the oxidation of dihydroorotate were originally identified in anaerobic bacteria 1 and mammalian cells 2 establishing the reaction as an obligatory step in the de novo synthesis of the pyrimidine heterocyclic ring. Constitutive enzymes responsible for catalyzing this step have been extensively investigated in prokaryotes 3 and fungi. 4 Although the molecular and catalytic properties of the enzyme differ depending on the source, biosynthetic dihydroorotate dehydrogenase (E.C. 1.3.3.15) is a mitochondrial enzyme in eukaryotic cells e'7 The oxidation of the substrate may be linked to reduction of components of the mitochondrial respiratory chain through quinones which are the primary electron acceptors for the enzyme purified as described in this article. The enzyme is a lipoprotein which can be isolated in catalytically active form from the mitochondrial membrane with nonionic detergents. Special treatment is required to prevent reaggregation or complete inactivation of the enzyme. Only dihydroorotate (2.5-4 mM) is capable of full protection of the isolated enzyme during purification. 6 Pyridine nucleotides cannot serve as electron acceptors for the fungal enzyme in situ nor can molecular oxygen serve as a primary electron acceptor for the isolated enzyme. 1 I. Lieberman and A. Kornberg, Biochim. Biophys. Acta 12, 223 (1953). 2 R. Wu and D. W. Wilson, J. Biol. Chem. 223, 195 (1956). 3 W. H. Taylor, M. L. Taylor, and D. F. Eames, J. Bacteriol. 91, 2251 (1966), 4 R. W. Miller, Arch. Biochem. Biophys. 146, 256 (1971). 5 See properties o f enzyme for consideration o f reactivity with oxygen. R. W. Miller, Can. J. Biochem. 53, 1288 (1975). r j. j. Chen and M. E. Jones, Arch. Biochem. Biophys. 176, 82 (1976).
M E T H O D S IN E N Z Y M O L O G Y , V O L . L I
Copyright ~) 1978by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181951