- Email: [email protected]
Direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate decarboxylase
ANALYTICAL
BIOCHEMISTRY
19
1,365-369
(1990)
Direct Spectrophotometric Phosphoribosyltransferase Decarboxylase’ Keith
Shostak,*
*Department iDepartment
Received
March
Richard
of Biochemistry, of Biochemistry,
I. Christopherson,-f University University
Assays for Orotate and Orotidylate and Mary
Ellen
Jones*v2
of North Carolina at Chapel Hill, Chapel Hill, North of Sydney, Sydney, NS W, 2006, Australia
Carolina
27599, and
12,199O
New sensitive and direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate5’-monophosphate (OMP) decarboxylase are described. The assays utilize a thioketone derivative of orotate (4thio-6-carboxyuracil) which is converted into 4-thioOMP by the transferase in the presence of phosphoribosyl pyrophosphate. 4-Thio-OMP is subsequently decarboxylated to I-thio-UMP by OMP decarboxylase. A novel, efficient synthesis of thioorotate is described. Unlike the natural substrates, the interconversion of the thioketone derivatives yields large spectral changes in the near-visible absorption region. Orotate phosphoribosyltransferase is assayed at 333 nm with a molar extinction coefficient of 16,306 M-’ cm-’ for the conversion of thioorotate to either 4-thio-OMP or 4thio-UMP. Orotidylate decarboxylase is assayed at 365 nm with a molar extinction coefficient of 3350 M-’ cm-’ for the conversion of I-thio-OMP to 4-thio-UMP. Another advantage of these substrates is that they bind less tightly to orotate phosphoribosyltransferase and OMP decarboxylase than orotate or OMP, respectively. Thus, the initial rates of substrate conversion to product are readily measurable near the K,,, values for the thioketone substrates. The ability to follow the reactions directly permits the rapid determination of K,,, values for the thioketone substrates and K,values for inhibitOrS of the enzymes. 0 1990 Academic press, k.
date (1). The last two enzymes of the pathway are orotate phosphoribosyltransferase (EC 2.4.2.10) and OMP3 decarboxylase (EC 4.1.1.23) which respectively catalyze reactions orotate
+ PP-ribose-P OMP + UMP
M8+ w
OMP
+ CO,
+ PPi
111 PI
UMP is synthesized by a six-step pathway in all living organisms capable of de novo UMP synthesis studied to
In mammals, the enzymes exist as part of a single, bifunctional protein known as UMP synthase (2). In fungi and bacteria, the two activities exist as separate proteins (3). Although several fixed-point assays have been developed for these enzymes (4-6), only one direct assay is currently in use (7). Lieberman et al. have shown that the transferase activity can be measured in the presence of decarboxylase by monitoring the decrease in absorbance at 295 nm as orotate is converted to UMP. Likewise, the conversion of OMP to UMP by the decarboxylase was followed by the decrease in absorbance at 285 nm. The assays are not very sensitive with molar extinction coefficients of 3950 M-r cm-’ for the transferase and 1650 M-’ cm-’ for the decarboxylase assays. These two spectrophotometric assays are useful for systems where (i) activity levels are high such that background absorption from DNA and proiein do not interfere with the uv absorption changes and (ii) the K,,, values for orotate and OMP are high enough to allow a measurable change in absorbance for assays in the K,,, range. In this study, 4-thioketo analogs of the physiological
r This work was supported by NIH grant CA-09156, and the Australian Research Council. ’ To whom correspondence should
3 Abbreviations used: OMP, orotidine 5’-monophosphate; PRPP, 5-phosphoribosyl 1-pyrophosphate; PPi, inorganic pyrophosphate; PEI, polyethyleneimine; Mea, 2-[N-morpholino]ethanesulfonic acid; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid.
0003-2697/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Grant GM-34539, National Health be addressed.
NC1 training and Medical
365 Inc. reserved.
366
SHOSTAK,
CHRISTOPHERSON,
substrates were synthesized in order to exploit the high extinction coefficients and red spectral shifts exhibited by the replacement of a keto group with a thioketone (8). The starting material, thioorotic acid (4-thio-6-carboxyuracil), is not commercially available at this time. The compound was originally synthesized by Daves et al. and involved six steps (9). However, an efficient and rapid synthesis is reported here using phosphorus pentasulfide as a sulfur donor. Orotate phosphoribosyltransferase can then be used to convert 4-thioorotate to 4-thio-OMP, the substrate for the OMP decarboxylase assay. The spectral assays reported here are direct so that not only the initial velocity of the reaction is immediately known, but also the spectral changes upon substrate conversion to product are in the near-visible region so crude extracts are easily assayed. The absorbance changes are large enough in relation to the thioketone K,,, values for rapid determination of kinetic constants. Finally, the assays are inexpensive as compared to the radiolabeled methods currently in use. MATERIALS
AND
METHODS
Anhydrous erotic acid, yeast orotate phosphoribosyltransferase, yeast orotidylate decarboxylase, OMP, sodium PRPP, yeast inorganic pyrophosphatase, and 4thio-UMP were purchased from Sigma. Phosphorus pentasulfide was purchased from Aldrich. PEI cellulose plates were obtained from Brinkmann. Kinetic studies were performed with either pure yeast orotidylate decarboxylase (Bell, J. B. and Jones, M. E., manuscript in preparation) or an ammonium sulfate precipitate from human placenta containing UMP synthase (10). Spectroscopic studies and assays were performed on a Milton Roy Spectronic 3000 diode array spectrophotometer. Synthesis of thioorotic acid. In an oil bath, anhydrous erotic acid (200 mg) was dissolved in 20 ml warm pyridine with stirring. Phosphorus pentasulfide (860 mg) was then added. The mixture was stirred and gradually heated to 90°C over a period of 30 min. Stirring and heating at 90°C were continued for a further 30 min. After cooling, the pyridine solution was decanted from the undissolved residue at the bottom of the reaction vessel and the decanted solution concentrated in a rotary evaporator at 40°C until dry. The dry residue was then stirred with 10% hydrochloric acid (12 ml) for 15 min at room temperature. The precipitated product was filtered, washed with a small amount of water and ethanol, and dried to yield an apricot orange crystalline solid (150 mg, 65% yield). The precipitated product typically contains about 10% unreacted orotate which is easily removed by HPLC using a C-18 reverse-phase column with 50 mM ammonium formate, pH 5.5, as solvent. The separation was monitored at 280 nm where thioorotate elutes immediately after orotate. The pure
AND
JONES
product had a molar extinction coefficient of 16,000 M-l cm-’ at 350 nm between pH 3 and 7. NMR spectra of thioorotate were obtained with a Bruker AMX 400 wide-bore spectrometer at 400 MHz for ‘H and 100 MHz for 13C. ‘H NMR (D,O) 6 6.835 (H5). 13C NMR (D,O containing 20% (w/v) NaOD) 6 193.1, (C4), 151.2 (C2), 143.8 (C6), 102.0 (C5), 169 (carboxy1 C at low intensity). Assignments for the 13C NMR spectrum were made by comparison with those for uracil, tetramethylthiourea, and benzoic acid (11). Highresolution mass spectra were obtained using a KratosMS902 with a VG 3D8 console update, direct insertion probe at 200°C source temperature, 8-kV acceleration voltage, and 70-eV ionization energy. Observed m/z 171.9975 (mean of five readings with zero standard error to four decimal places; M, 8.1%), calculated mass for C,H,N,O,S, 171.9942. A 2OOqL reaction was set Synthesis of 4-thio-OMP. up containing the following: 50 mM Tris-HCl, pH. 8.0, 120 mM sodium PRPP, 100 mM MgCl,, 50 units yeast orotate phosphoribosyltransferase, 50 units yeast inorganic pyrophosphatase, 25 mM thioorotate acid. The reaction mixture was incubated in the dark at 30°C for 3 h. The sample was then boiled for 2 min and centrifuged. The product was purified from the reaction mixture by HPLC on a Whatman Partisil 10 SAX column using 0.4 M ammonium bicarbonate, pH 7.4, as the solvent. For routine assays of pure yeast OMP decarboxylase, it was found that purification of 4-thio-OMP from the reaction mix is probably not essential because the kinetic constants obtained with substrate from the reaction mix are identical with those obtained when purified substrate was used. Pure 4-thio-OMP has a molar extinction coefficient of 23,000 M-’ cm-’ at 335 nm between pH 3 and 7. The concentration of 4-thio-OMP in the reaction mix was determined by noting the change in absorbance at 365 nm as 4-thio-OMP was completely converted to 4-thio-UMP by the decarboxylase. The extinction coefficient for this change is 3350 M-’ cm-‘. Enzyme assays. All assays were performed in a final volume of 0.5 ml at 23°C and were linear with enzyme concentration. The following molar extinction coefficients were used in the concentration determinations of substrates and products: 3950 M-l cm-’ for erotic acid to UMP at 295 nm (7), 1650 M-l cm-’ for OMP to UMP at 285 nm (7), 10,300 M-’ cm-’ for thioorotate to either 4-thio-OMP or 4-thio-UMP at 333 nm, and 3350 M-’ cm-’ for 4-thio-OMP to 4-thio-UMP at 365 nm. Orotate phosphoribosyltransferase was typically assayed in a buffer mix containing 250 pM PRPP and 5 mM MgCl,. Buffer and substrate conditions varied and are described in the text. RESULTS
AND
DISCUSSION
Spectra of thio substrates and products. Figure 1 shows the spectral changes as thioorotic acid is con-
SPECTROPHOTOMETRIC
ASSAYS
FOR
25
URIDINEd’-MONOPHOSPHATE
367
SYNTHASE
from N3 of the pyrimidine ring. For thioorotate, the spectral changes upon titration were not large enough to provide an accurate determination of the pK,. However, the pK, was estimated from the data to fall between 8.0 and 8.5. The acidity of the thioketone pyrimidine rings precludes the use of the given extinction coefficients in assays at high pH. We have found that the extinction coefficients are valid for assays performed between pH 4.0 and 7.0.
Separation
WAVELENGTH
(nm)
FIG. 1. Absorption spectra of equimolar amounts of thioorotate (---), 4-thio-OMP (-), and 4-thio-UMP ( * . * 1. The spectra were generated by the complete enzymatic conversion of 30 gM thioorotate to 4-thio-OMP by orotate phosphoribosyltransferase. The isosbestic point for this conversion is observed at 351 nm. A catalytic amount of OMP decarboxylase was added to the sample resulting in the 4-thioUMP spectra. The spectra were obtained in 20 mM sodium Mes, pH 6.0, and are representative of trials at different pH values up to 7.0 in which the background absorption due to the buffer, proteins, etc. are subtracted.
verted to 4-thio-OMP by orotate phosphoribosyltransferase, and when 4-thio-OMP is subsequently decarboxylated to 4-thio-UMP by orotidylate decarboxylase. The spectra of the thioketones closely resemble those of the natural substrates and products except that the extinction coefficients are approximately twofold higher and the thioketone spectra are shifted to the red by about 70 nm. The wavelengths for enzymatic assay were selected in the areas exhibiting the largest absorption change upon conversion of substrate to product. For orotate phosphoribosyltransferase, this wavelength is 333 nm with a molar extinction coefficient of 10,300 M-l cm-l for the conversion of thioorotate to either 4-thioOMP in the absence of decarboxylase or 4-thio-UMP in the presence of decarboxylase. The extinction coefficient is independent of which product is formed because the assay wavelength is at the isosbestic point for the conversion of 4-thio-OMP to 4-thio-UMP. For orotidylate decarboxylase, the assay wavelength is 365 nm with a molar extinction coefficient of 3350 M-l cm-’ for the conversion of 4-thio-OMP to 4-thio-UMP. The effect of pH on the spectra of substrates and products was investigated because ionization of the pyrimidine ring would change the extinction coefficient values given for the assays. An apparent pK, of 8.2 was found by spectrophometric titration of 4-thio-UMP. The pK, probably reflects the dissociation the proton
of substrates
and products
on PEI-cellu-
lose. Thioorotate, 4-thio-OMP, and 4-thio-UMP are readily separated on PEI-cellulose thin-layer plates previously developed with 0.2 M LiCl. Enzymatically prepared samples and standards were spotted on 20-cm plates 2.5 cm from the bottom. The plates were developed for 5 min in 0.2 M LiCl and then immediately transferred to a tank containing 1.5 M LiCl and developed for another 55 min. Spots were visualized under an ultraviolet lamp where 4-thio-OMP, thioorotate, and 4thio-UMP had R, values of 0.41,0.50, and 0.60, respectively. No other spots were observed in the enzyme-generated samples.
Comparison of OMP decarboxyluse kinetic constants with 4-thio-OMP and OMP. A Lineweaver-Burk plot comparing the decarboxylation of OMP and 4-thioOMP by the yeast enzyme shows that 4-thio-OMP is decarboxylated at about half the rate of OMP exhibits a K, value four- to fivefold higher than the natural substrate (Fig. 2) The K, of 6 I.IM for OMP agrees well with the K,,, determined by Umezu et al. of 5 PM under nearly identical assay conditions in 100 mM phosphate (12). In these assays, phosphate acts as a competitive inhibitor, increasing the apparent K,.,, value while leaving V,, un-
OMP
-0.10
0.00 i/[S]
FIG. 2.
0.10
0.20
uM
Lineweaver-Burk plots of yeast OMP decarboxylase activity using OMP (w) or I-thio-OMP (0) as substrate. The assay concltions were in 100 mM potassium phosphate, pH 6.0, 1 mM 2-mercaptoethanol at 23°C. The apparent Km values for OMP and 4-thio-OMP under these conditions are 6 and 29 pM, respectively. The apparent V,, values are 46 and 21 rmollmin/mg protein, respectively.
368
SHOSTAK, TABLE OMP
Decarboxylase
OMPcd 4-thio-OMPC Phosphate 6-aza-UMP
vmax 7.3 x 1o-4 2.7 x 1O-3 -
Kinetic
Constants Yeastb
Apparent K,,, or Ki
V,,
Apparent K,,, or Ki
9.5 PM 511 nM
40 21 -
3.5 pMe 20 pM 20 mM 980 nM
o The human enzyme was assayed in 1 mM 2-mercaptoethanol and 200 mM potassium phosphate, pH 7.0, at 23°C. All values were obtained using an ammonium sulfate fraction of homogenized placenta (10). b The yeast enzyme was assayed in 1 mM 2-mercaptoethanol and 50 mM potassium phosphate, pH 6.0, at 23’C. All values were obtained with pure enzyme. ’ The apparent V,. value is expressed in units of amol/min/mg protein. d The values given here for V,, are actually those for the specific activity determined with 40 PM OMP using both the spectrophotometric (7) and radioactive (5) assays. e The K,,, value for OMP, as obtained in Fig. 2 where 166 mM phosphate buffer was used, was corrected to the value expected at 50 mM phosphate using the competitive inhibition constant for phosphate of
20mM.
The spectrophotometric assay using 4-thiois sensitive enough to obtain a K, value in the absence of phosphate. A Ki value for phosphate of 20 mM was found for the yeast enzyme using 4-thio-OMP as substrate (Table 1). For routine assay work with the yeast decarboxylase, the presence of 50 mM phosphate is suggested because most of the previous studies with the enzyme have been performed in this buffer (13) and the K, value for 4-thio-OMP (20 PM) is more easily obtained for inhibition studies (Table 1). 4-thio-OMP is also decarboxylated by human UMP synthase. The assays were performed in 200 mM phosphate as the enzyme from mammalian systems tends to bind nucleotide substrates and inhibitors several fold more tightly than OMP decarboxylase from yeast (6,13). The specific activity of the enzyme in 200 mM phosphate is about fourfold higher with 4-thio-OMP than the natural substrate, OMP. The apparent K, under these conditions was 9.5 PM (Table 1). The Kj of phosphate could not be determined with this assay because the assay is not sensitive enough to determine K,,, values less than 5 PM. However, the high specific enzyme activity observed with 4-thio-OMP should prove useful in quantitating activity of mammalian UMP synthase in homogenates or purified fractions. Effect of pH on the OMP decarboxylation assay. The effect of pH on specific activity of yeast OMP decarboxylase was examined to determine if the slower decarboxylation rate of 4-thio-OMP as compared to OMP was affected.
OMP
AND JONES
1
Human“ Substrate or inhibitor
CHRISTOPHERSON,
0-l
3.5
,
4.3
.
,
5.5
,
63
7! 5
PH
FIG. 3. pH profile of yeast OMP decarboxylase specific activity using OMP (m) or 4-thio-OMP (Cl) as substrate. Activity was measured at 23°C in the presence of either 40 PM OMP or 166 PM 4-thioOMP. Assays were performed in a 50 rnrvfsodium acetate-Mes buffer system.
due to an alteration of the pH optimum of the enzyme. Specific activity profiles of 4-thio-OMP and OMP demonstrate that the pH optimum is approximately 5.5 for either substrate (Fig. 3). The relatively similar profiles may suggest that 4-thio-OMP is decarboxylated by the same mechanism as OMP. Comparison of orotate phosphoribosyltransferase kinetic constants with those of orotate and thioorotate. Lineweaver-Burk plots comparing orotate and thioorotate as substrates for the yeast orotate phosphoribosyltransferase reaction are shown in Fig. 4. As with the
OA
-0.06
-0.04
-0.02
0.0
I /ISI
0.02
0.04
I 56
uM
FIG. 4. Lineweaver-Burk plots of yeast orotate phosphoribosyltransferase activity using orotate (w) or thioorotate (Cl) as substrates in the presence of 1 unit OMP decarboxylase. In the absence of added decarboxylase, thioorotate (A) exhibited similar spectral changes describing the initial rates. Assays were performed in 20 mu sodium Bes, pH 7.0, at 23%
SPECTROPHOTOMETRIC
ASSAYS
FOR
URIDINEd’-MONOPHOSPHATE
yeast OMP decarboxylase, the natural substrate binds more tightly (K, = 15.4 FM) than the thioketone substrate (K, = 23.5 PM). A limitation of thioorotate as a substrate is that it undergoes phosphoribosyl transfer at 8-9% of the rate with orotate as substrate. Furthermore, the assay is only functional up to pH 7.0; the pH optimum of yeast orotate phosphoribosyltransferase with orotate as substrate is 8.5 to 9 (12). With human UMP synthase, activity was detected with the thioorotate substrate. However, the activity in the ammonium sulfate fractions was too low to accurately quantitate. Thus, it would appear that thioorotate is a poor substrate for assays of crude preparations of human extracts, and possibly in other animal extracts. Although the reaction is slow, there are advantages of the thioorotate assay over the orotate assay. The thioorotate assay does not require the addition of exogenous OMP decarboxylase to the assay mix. The initial rate and absorbance change with an extinction coefficient of 10,300 M-’ cm-’ at 333 nm are independent of the amount of 4-thio-UMP formed from 4-thio-OMP (Fig. 4). This is due to the selection of an assay wavelength at the isosbestic point in the conversion of 4thio-OMP to 4-thio-UMP (Fig. 1). Since these compounds apparently have the same extinction coefficient, the absorbance at the isosbestic point will reflect the concentration of either product equally. The spectrophotometric assay utilizing orotate requires the addition of excess decarboxylase because the absorbance decrease measured at 295 nm partially depends on the rapid conversion of OMP to UMP. Another advantage is that the thioorotate assay is inherently more sensitive than the orotate assay because the extinction coefficient
369
SYNTHASE
is about three times greater. Finally, the near-visible absorption changes could make this compound useful for spectrophotometric mechanism studies requiring stoichiometric concentrations of enzyme. ACKNOWLEDGMENT We thank Dr. Leo Phillips mass spectroscopy.
for ‘H and “C NMR
and high-resolution
REFERENCES 1. Jones,
M. E. (1980)
Annu. Reu. B&hem.
2. McClard, R. W., Black, M. J., Livingstone, (1980) Biochemistry 19,4699-4706. 3. O’Donovan, G. A., and Neuhard, J. (1970)
49,253-279. L. R., and Jones,
Bacterid
M. E.
Reu. 34,278-
343. 4. Rogers,
L. E. (1973)
Amer. J. Clin. Pathol. 69,31-35.
5. Prabhakararao, K., and Jones, M. E. (1975) Anal. Biochem. 451-467. 6. Traut, T. W., and Jones, M. E. (1977) Biochem. Pharmacol. 2291-2296. 7. Lieberman,
I., Kornberg,
A., and
Simms,
Z., and
Shugar,
E. S. (1955)
69, 26,
J. Biol.
Chem. 215,403-415. 8. Psoda,
A., Kazimierczuk,
D. (1974)
J. Amer.
Chem. Sot. 96,6832-6839. 9. Daves,
G. D., Baiocchi,
F., Robins,
R. K., and Cheng,
C. C. (1961)
J. Amer. Chem. See. 26,2755-2763. 10. Livingstone, L. R., and Jones, M. E. (1987) J. Biof. Chem. 262, 15,726-15,733. 11. Johnson, L. F., and Jankowski, W. C. (1972) Carbon-13 NMR Spectra. A Collection of Assigned, Coded and Indexed Spectra, Wiley, New York. 12. Umezu,
K., Amaya,
T., Yoshimoto,
A., and Tomita,
K. (1971)
J.
Biochem. 70, 249-262. 13. Levine,
H. L., Brody,
chemistry 19,4993-4999.
R. S., and Westheimer,
F. H. (1980)
Bio-
BIOCHEMISTRY
19
1,365-369
(1990)
Direct Spectrophotometric Phosphoribosyltransferase Decarboxylase’ Keith
Shostak,*
*Department iDepartment
Received
March
Richard
of Biochemistry, of Biochemistry,
I. Christopherson,-f University University
Assays for Orotate and Orotidylate and Mary
Ellen
Jones*v2
of North Carolina at Chapel Hill, Chapel Hill, North of Sydney, Sydney, NS W, 2006, Australia
Carolina
27599, and
12,199O
New sensitive and direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate5’-monophosphate (OMP) decarboxylase are described. The assays utilize a thioketone derivative of orotate (4thio-6-carboxyuracil) which is converted into 4-thioOMP by the transferase in the presence of phosphoribosyl pyrophosphate. 4-Thio-OMP is subsequently decarboxylated to I-thio-UMP by OMP decarboxylase. A novel, efficient synthesis of thioorotate is described. Unlike the natural substrates, the interconversion of the thioketone derivatives yields large spectral changes in the near-visible absorption region. Orotate phosphoribosyltransferase is assayed at 333 nm with a molar extinction coefficient of 16,306 M-’ cm-’ for the conversion of thioorotate to either 4-thio-OMP or 4thio-UMP. Orotidylate decarboxylase is assayed at 365 nm with a molar extinction coefficient of 3350 M-’ cm-’ for the conversion of I-thio-OMP to 4-thio-UMP. Another advantage of these substrates is that they bind less tightly to orotate phosphoribosyltransferase and OMP decarboxylase than orotate or OMP, respectively. Thus, the initial rates of substrate conversion to product are readily measurable near the K,,, values for the thioketone substrates. The ability to follow the reactions directly permits the rapid determination of K,,, values for the thioketone substrates and K,values for inhibitOrS of the enzymes. 0 1990 Academic press, k.
date (1). The last two enzymes of the pathway are orotate phosphoribosyltransferase (EC 2.4.2.10) and OMP3 decarboxylase (EC 4.1.1.23) which respectively catalyze reactions orotate
+ PP-ribose-P OMP + UMP
M8+ w
OMP
+ CO,
+ PPi
111 PI
UMP is synthesized by a six-step pathway in all living organisms capable of de novo UMP synthesis studied to
In mammals, the enzymes exist as part of a single, bifunctional protein known as UMP synthase (2). In fungi and bacteria, the two activities exist as separate proteins (3). Although several fixed-point assays have been developed for these enzymes (4-6), only one direct assay is currently in use (7). Lieberman et al. have shown that the transferase activity can be measured in the presence of decarboxylase by monitoring the decrease in absorbance at 295 nm as orotate is converted to UMP. Likewise, the conversion of OMP to UMP by the decarboxylase was followed by the decrease in absorbance at 285 nm. The assays are not very sensitive with molar extinction coefficients of 3950 M-r cm-’ for the transferase and 1650 M-’ cm-’ for the decarboxylase assays. These two spectrophotometric assays are useful for systems where (i) activity levels are high such that background absorption from DNA and proiein do not interfere with the uv absorption changes and (ii) the K,,, values for orotate and OMP are high enough to allow a measurable change in absorbance for assays in the K,,, range. In this study, 4-thioketo analogs of the physiological
r This work was supported by NIH grant CA-09156, and the Australian Research Council. ’ To whom correspondence should
3 Abbreviations used: OMP, orotidine 5’-monophosphate; PRPP, 5-phosphoribosyl 1-pyrophosphate; PPi, inorganic pyrophosphate; PEI, polyethyleneimine; Mea, 2-[N-morpholino]ethanesulfonic acid; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid.
0003-2697/90 Copyright All rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Grant GM-34539, National Health be addressed.
NC1 training and Medical
365 Inc. reserved.
366
SHOSTAK,
CHRISTOPHERSON,
substrates were synthesized in order to exploit the high extinction coefficients and red spectral shifts exhibited by the replacement of a keto group with a thioketone (8). The starting material, thioorotic acid (4-thio-6-carboxyuracil), is not commercially available at this time. The compound was originally synthesized by Daves et al. and involved six steps (9). However, an efficient and rapid synthesis is reported here using phosphorus pentasulfide as a sulfur donor. Orotate phosphoribosyltransferase can then be used to convert 4-thioorotate to 4-thio-OMP, the substrate for the OMP decarboxylase assay. The spectral assays reported here are direct so that not only the initial velocity of the reaction is immediately known, but also the spectral changes upon substrate conversion to product are in the near-visible region so crude extracts are easily assayed. The absorbance changes are large enough in relation to the thioketone K,,, values for rapid determination of kinetic constants. Finally, the assays are inexpensive as compared to the radiolabeled methods currently in use. MATERIALS
AND
METHODS
Anhydrous erotic acid, yeast orotate phosphoribosyltransferase, yeast orotidylate decarboxylase, OMP, sodium PRPP, yeast inorganic pyrophosphatase, and 4thio-UMP were purchased from Sigma. Phosphorus pentasulfide was purchased from Aldrich. PEI cellulose plates were obtained from Brinkmann. Kinetic studies were performed with either pure yeast orotidylate decarboxylase (Bell, J. B. and Jones, M. E., manuscript in preparation) or an ammonium sulfate precipitate from human placenta containing UMP synthase (10). Spectroscopic studies and assays were performed on a Milton Roy Spectronic 3000 diode array spectrophotometer. Synthesis of thioorotic acid. In an oil bath, anhydrous erotic acid (200 mg) was dissolved in 20 ml warm pyridine with stirring. Phosphorus pentasulfide (860 mg) was then added. The mixture was stirred and gradually heated to 90°C over a period of 30 min. Stirring and heating at 90°C were continued for a further 30 min. After cooling, the pyridine solution was decanted from the undissolved residue at the bottom of the reaction vessel and the decanted solution concentrated in a rotary evaporator at 40°C until dry. The dry residue was then stirred with 10% hydrochloric acid (12 ml) for 15 min at room temperature. The precipitated product was filtered, washed with a small amount of water and ethanol, and dried to yield an apricot orange crystalline solid (150 mg, 65% yield). The precipitated product typically contains about 10% unreacted orotate which is easily removed by HPLC using a C-18 reverse-phase column with 50 mM ammonium formate, pH 5.5, as solvent. The separation was monitored at 280 nm where thioorotate elutes immediately after orotate. The pure
AND
JONES
product had a molar extinction coefficient of 16,000 M-l cm-’ at 350 nm between pH 3 and 7. NMR spectra of thioorotate were obtained with a Bruker AMX 400 wide-bore spectrometer at 400 MHz for ‘H and 100 MHz for 13C. ‘H NMR (D,O) 6 6.835 (H5). 13C NMR (D,O containing 20% (w/v) NaOD) 6 193.1, (C4), 151.2 (C2), 143.8 (C6), 102.0 (C5), 169 (carboxy1 C at low intensity). Assignments for the 13C NMR spectrum were made by comparison with those for uracil, tetramethylthiourea, and benzoic acid (11). Highresolution mass spectra were obtained using a KratosMS902 with a VG 3D8 console update, direct insertion probe at 200°C source temperature, 8-kV acceleration voltage, and 70-eV ionization energy. Observed m/z 171.9975 (mean of five readings with zero standard error to four decimal places; M, 8.1%), calculated mass for C,H,N,O,S, 171.9942. A 2OOqL reaction was set Synthesis of 4-thio-OMP. up containing the following: 50 mM Tris-HCl, pH. 8.0, 120 mM sodium PRPP, 100 mM MgCl,, 50 units yeast orotate phosphoribosyltransferase, 50 units yeast inorganic pyrophosphatase, 25 mM thioorotate acid. The reaction mixture was incubated in the dark at 30°C for 3 h. The sample was then boiled for 2 min and centrifuged. The product was purified from the reaction mixture by HPLC on a Whatman Partisil 10 SAX column using 0.4 M ammonium bicarbonate, pH 7.4, as the solvent. For routine assays of pure yeast OMP decarboxylase, it was found that purification of 4-thio-OMP from the reaction mix is probably not essential because the kinetic constants obtained with substrate from the reaction mix are identical with those obtained when purified substrate was used. Pure 4-thio-OMP has a molar extinction coefficient of 23,000 M-’ cm-’ at 335 nm between pH 3 and 7. The concentration of 4-thio-OMP in the reaction mix was determined by noting the change in absorbance at 365 nm as 4-thio-OMP was completely converted to 4-thio-UMP by the decarboxylase. The extinction coefficient for this change is 3350 M-’ cm-‘. Enzyme assays. All assays were performed in a final volume of 0.5 ml at 23°C and were linear with enzyme concentration. The following molar extinction coefficients were used in the concentration determinations of substrates and products: 3950 M-l cm-’ for erotic acid to UMP at 295 nm (7), 1650 M-l cm-’ for OMP to UMP at 285 nm (7), 10,300 M-’ cm-’ for thioorotate to either 4-thio-OMP or 4-thio-UMP at 333 nm, and 3350 M-’ cm-’ for 4-thio-OMP to 4-thio-UMP at 365 nm. Orotate phosphoribosyltransferase was typically assayed in a buffer mix containing 250 pM PRPP and 5 mM MgCl,. Buffer and substrate conditions varied and are described in the text. RESULTS
AND
DISCUSSION
Spectra of thio substrates and products. Figure 1 shows the spectral changes as thioorotic acid is con-
SPECTROPHOTOMETRIC
ASSAYS
FOR
25
URIDINEd’-MONOPHOSPHATE
367
SYNTHASE
from N3 of the pyrimidine ring. For thioorotate, the spectral changes upon titration were not large enough to provide an accurate determination of the pK,. However, the pK, was estimated from the data to fall between 8.0 and 8.5. The acidity of the thioketone pyrimidine rings precludes the use of the given extinction coefficients in assays at high pH. We have found that the extinction coefficients are valid for assays performed between pH 4.0 and 7.0.
Separation
WAVELENGTH
(nm)
FIG. 1. Absorption spectra of equimolar amounts of thioorotate (---), 4-thio-OMP (-), and 4-thio-UMP ( * . * 1. The spectra were generated by the complete enzymatic conversion of 30 gM thioorotate to 4-thio-OMP by orotate phosphoribosyltransferase. The isosbestic point for this conversion is observed at 351 nm. A catalytic amount of OMP decarboxylase was added to the sample resulting in the 4-thioUMP spectra. The spectra were obtained in 20 mM sodium Mes, pH 6.0, and are representative of trials at different pH values up to 7.0 in which the background absorption due to the buffer, proteins, etc. are subtracted.
verted to 4-thio-OMP by orotate phosphoribosyltransferase, and when 4-thio-OMP is subsequently decarboxylated to 4-thio-UMP by orotidylate decarboxylase. The spectra of the thioketones closely resemble those of the natural substrates and products except that the extinction coefficients are approximately twofold higher and the thioketone spectra are shifted to the red by about 70 nm. The wavelengths for enzymatic assay were selected in the areas exhibiting the largest absorption change upon conversion of substrate to product. For orotate phosphoribosyltransferase, this wavelength is 333 nm with a molar extinction coefficient of 10,300 M-l cm-l for the conversion of thioorotate to either 4-thioOMP in the absence of decarboxylase or 4-thio-UMP in the presence of decarboxylase. The extinction coefficient is independent of which product is formed because the assay wavelength is at the isosbestic point for the conversion of 4-thio-OMP to 4-thio-UMP. For orotidylate decarboxylase, the assay wavelength is 365 nm with a molar extinction coefficient of 3350 M-l cm-’ for the conversion of 4-thio-OMP to 4-thio-UMP. The effect of pH on the spectra of substrates and products was investigated because ionization of the pyrimidine ring would change the extinction coefficient values given for the assays. An apparent pK, of 8.2 was found by spectrophometric titration of 4-thio-UMP. The pK, probably reflects the dissociation the proton
of substrates
and products
on PEI-cellu-
lose. Thioorotate, 4-thio-OMP, and 4-thio-UMP are readily separated on PEI-cellulose thin-layer plates previously developed with 0.2 M LiCl. Enzymatically prepared samples and standards were spotted on 20-cm plates 2.5 cm from the bottom. The plates were developed for 5 min in 0.2 M LiCl and then immediately transferred to a tank containing 1.5 M LiCl and developed for another 55 min. Spots were visualized under an ultraviolet lamp where 4-thio-OMP, thioorotate, and 4thio-UMP had R, values of 0.41,0.50, and 0.60, respectively. No other spots were observed in the enzyme-generated samples.
Comparison of OMP decarboxyluse kinetic constants with 4-thio-OMP and OMP. A Lineweaver-Burk plot comparing the decarboxylation of OMP and 4-thioOMP by the yeast enzyme shows that 4-thio-OMP is decarboxylated at about half the rate of OMP exhibits a K, value four- to fivefold higher than the natural substrate (Fig. 2) The K, of 6 I.IM for OMP agrees well with the K,,, determined by Umezu et al. of 5 PM under nearly identical assay conditions in 100 mM phosphate (12). In these assays, phosphate acts as a competitive inhibitor, increasing the apparent K,.,, value while leaving V,, un-
OMP
-0.10
0.00 i/[S]
FIG. 2.
0.10
0.20
uM
Lineweaver-Burk plots of yeast OMP decarboxylase activity using OMP (w) or I-thio-OMP (0) as substrate. The assay concltions were in 100 mM potassium phosphate, pH 6.0, 1 mM 2-mercaptoethanol at 23°C. The apparent Km values for OMP and 4-thio-OMP under these conditions are 6 and 29 pM, respectively. The apparent V,, values are 46 and 21 rmollmin/mg protein, respectively.
368
SHOSTAK, TABLE OMP
Decarboxylase
OMPcd 4-thio-OMPC Phosphate 6-aza-UMP
vmax 7.3 x 1o-4 2.7 x 1O-3 -
Kinetic
Constants Yeastb
Apparent K,,, or Ki
V,,
Apparent K,,, or Ki
9.5 PM 511 nM
40 21 -
3.5 pMe 20 pM 20 mM 980 nM
o The human enzyme was assayed in 1 mM 2-mercaptoethanol and 200 mM potassium phosphate, pH 7.0, at 23°C. All values were obtained using an ammonium sulfate fraction of homogenized placenta (10). b The yeast enzyme was assayed in 1 mM 2-mercaptoethanol and 50 mM potassium phosphate, pH 6.0, at 23’C. All values were obtained with pure enzyme. ’ The apparent V,. value is expressed in units of amol/min/mg protein. d The values given here for V,, are actually those for the specific activity determined with 40 PM OMP using both the spectrophotometric (7) and radioactive (5) assays. e The K,,, value for OMP, as obtained in Fig. 2 where 166 mM phosphate buffer was used, was corrected to the value expected at 50 mM phosphate using the competitive inhibition constant for phosphate of
20mM.
The spectrophotometric assay using 4-thiois sensitive enough to obtain a K, value in the absence of phosphate. A Ki value for phosphate of 20 mM was found for the yeast enzyme using 4-thio-OMP as substrate (Table 1). For routine assay work with the yeast decarboxylase, the presence of 50 mM phosphate is suggested because most of the previous studies with the enzyme have been performed in this buffer (13) and the K, value for 4-thio-OMP (20 PM) is more easily obtained for inhibition studies (Table 1). 4-thio-OMP is also decarboxylated by human UMP synthase. The assays were performed in 200 mM phosphate as the enzyme from mammalian systems tends to bind nucleotide substrates and inhibitors several fold more tightly than OMP decarboxylase from yeast (6,13). The specific activity of the enzyme in 200 mM phosphate is about fourfold higher with 4-thio-OMP than the natural substrate, OMP. The apparent K, under these conditions was 9.5 PM (Table 1). The Kj of phosphate could not be determined with this assay because the assay is not sensitive enough to determine K,,, values less than 5 PM. However, the high specific enzyme activity observed with 4-thio-OMP should prove useful in quantitating activity of mammalian UMP synthase in homogenates or purified fractions. Effect of pH on the OMP decarboxylation assay. The effect of pH on specific activity of yeast OMP decarboxylase was examined to determine if the slower decarboxylation rate of 4-thio-OMP as compared to OMP was affected.
OMP
AND JONES
1
Human“ Substrate or inhibitor
CHRISTOPHERSON,
0-l
3.5
,
4.3
.
,
5.5
,
63
7! 5
PH
FIG. 3. pH profile of yeast OMP decarboxylase specific activity using OMP (m) or 4-thio-OMP (Cl) as substrate. Activity was measured at 23°C in the presence of either 40 PM OMP or 166 PM 4-thioOMP. Assays were performed in a 50 rnrvfsodium acetate-Mes buffer system.
due to an alteration of the pH optimum of the enzyme. Specific activity profiles of 4-thio-OMP and OMP demonstrate that the pH optimum is approximately 5.5 for either substrate (Fig. 3). The relatively similar profiles may suggest that 4-thio-OMP is decarboxylated by the same mechanism as OMP. Comparison of orotate phosphoribosyltransferase kinetic constants with those of orotate and thioorotate. Lineweaver-Burk plots comparing orotate and thioorotate as substrates for the yeast orotate phosphoribosyltransferase reaction are shown in Fig. 4. As with the
OA
-0.06
-0.04
-0.02
0.0
I /ISI
0.02
0.04
I 56
uM
FIG. 4. Lineweaver-Burk plots of yeast orotate phosphoribosyltransferase activity using orotate (w) or thioorotate (Cl) as substrates in the presence of 1 unit OMP decarboxylase. In the absence of added decarboxylase, thioorotate (A) exhibited similar spectral changes describing the initial rates. Assays were performed in 20 mu sodium Bes, pH 7.0, at 23%
SPECTROPHOTOMETRIC
ASSAYS
FOR
URIDINEd’-MONOPHOSPHATE
yeast OMP decarboxylase, the natural substrate binds more tightly (K, = 15.4 FM) than the thioketone substrate (K, = 23.5 PM). A limitation of thioorotate as a substrate is that it undergoes phosphoribosyl transfer at 8-9% of the rate with orotate as substrate. Furthermore, the assay is only functional up to pH 7.0; the pH optimum of yeast orotate phosphoribosyltransferase with orotate as substrate is 8.5 to 9 (12). With human UMP synthase, activity was detected with the thioorotate substrate. However, the activity in the ammonium sulfate fractions was too low to accurately quantitate. Thus, it would appear that thioorotate is a poor substrate for assays of crude preparations of human extracts, and possibly in other animal extracts. Although the reaction is slow, there are advantages of the thioorotate assay over the orotate assay. The thioorotate assay does not require the addition of exogenous OMP decarboxylase to the assay mix. The initial rate and absorbance change with an extinction coefficient of 10,300 M-’ cm-’ at 333 nm are independent of the amount of 4-thio-UMP formed from 4-thio-OMP (Fig. 4). This is due to the selection of an assay wavelength at the isosbestic point in the conversion of 4thio-OMP to 4-thio-UMP (Fig. 1). Since these compounds apparently have the same extinction coefficient, the absorbance at the isosbestic point will reflect the concentration of either product equally. The spectrophotometric assay utilizing orotate requires the addition of excess decarboxylase because the absorbance decrease measured at 295 nm partially depends on the rapid conversion of OMP to UMP. Another advantage is that the thioorotate assay is inherently more sensitive than the orotate assay because the extinction coefficient
369
SYNTHASE
is about three times greater. Finally, the near-visible absorption changes could make this compound useful for spectrophotometric mechanism studies requiring stoichiometric concentrations of enzyme. ACKNOWLEDGMENT We thank Dr. Leo Phillips mass spectroscopy.
for ‘H and “C NMR
and high-resolution
REFERENCES 1. Jones,
M. E. (1980)
Annu. Reu. B&hem.
2. McClard, R. W., Black, M. J., Livingstone, (1980) Biochemistry 19,4699-4706. 3. O’Donovan, G. A., and Neuhard, J. (1970)
49,253-279. L. R., and Jones,
Bacterid
M. E.
Reu. 34,278-
343. 4. Rogers,
L. E. (1973)
Amer. J. Clin. Pathol. 69,31-35.
5. Prabhakararao, K., and Jones, M. E. (1975) Anal. Biochem. 451-467. 6. Traut, T. W., and Jones, M. E. (1977) Biochem. Pharmacol. 2291-2296. 7. Lieberman,
I., Kornberg,
A., and
Simms,
Z., and
Shugar,
E. S. (1955)
69, 26,
J. Biol.
Chem. 215,403-415. 8. Psoda,
A., Kazimierczuk,
D. (1974)
J. Amer.
Chem. Sot. 96,6832-6839. 9. Daves,
G. D., Baiocchi,
F., Robins,
R. K., and Cheng,
C. C. (1961)
J. Amer. Chem. See. 26,2755-2763. 10. Livingstone, L. R., and Jones, M. E. (1987) J. Biof. Chem. 262, 15,726-15,733. 11. Johnson, L. F., and Jankowski, W. C. (1972) Carbon-13 NMR Spectra. A Collection of Assigned, Coded and Indexed Spectra, Wiley, New York. 12. Umezu,
K., Amaya,
T., Yoshimoto,
A., and Tomita,
K. (1971)
J.
Biochem. 70, 249-262. 13. Levine,
H. L., Brody,
chemistry 19,4993-4999.
R. S., and Westheimer,
F. H. (1980)
Bio-