Purification and characterization of dihydropyrimidine dehydrogenase ...

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Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 31 ..... A273 : A454 is about 3.7 for dihydroorotate dehydrogenase.
Eur. J. Biochem. 155, 239-224 (1989) 8 FEBS 1989

Purification and characterization of dihydropyrimidine dehydrogenase from pig liver Beate PODSCHUN, Gudrun WAHLER and Klaus D. SCHNACKERZ Institutc of Physiological Chemistry, University of Wurzburg (Received February 22/June 19, 1989)

~

EJB 89 0212

Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100-fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE-cellulose and 2',5'-ADP - Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C-terminal 12-kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206-kDa molecule. The iron atoms are probably present in iron-sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5-aminouracil, 5-fluorouracil and NADPH. Pyrimidines are key monomers of nucleic acids and the selective inhibition of their catabolic pathway has been a strategy in designing antitumor, antimicrobial and potentially antiparasitic agents. One potential point of attack is the convcrsion of uracil to 5.6-dihydrouracil catalysed by dihydropyrimidine dehydrogenase (EC 1.3.1.2) which is the first reaction in the catabolic three-step sequence converting uracil via 5,6-dihydrouracil and N-carbamoyl-8-alanine into 8-alaninc. 0

0

ter activity in muscle and brain [ 5 ] . Using double-label techniques, it has clearly been shown that the three enzymes of the degradative pathway of uracil are also present in brain cytosol [6]. The main activity of dihydropyrimidine dehydrogenase is found in liver [7]. We report here the purification of dihydropyrimidine dehydrogenase from pig liver cytosol and its physicochemical characterization.

MATERIALS AND METHODS

Chemicah

In mammalian tissues this is the only pathway leading to the biosynthesis of 8-alanine [ 11. 8-Alanine (3-aminopropionic acid) is structurally related to 4-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system. Recently 8-alanine itself has been proposed to bc involved in synaptic transmission [2, 31. In addition /3alanine is efficiently taken up by some specialized brain cells (e. g. astrocytes) with a transport mechanism characteristic of a neurotransmitter [4]. P-Alanine-containing dipeptides, such as carnosine, anserine and N"-(b-alanyl)lysine have transmitCorrespondence to K. D. Schnackerz, Physiologisch-chemisches Institut der Universitat Wurzburg, KoellikerstraBe 2, D-8700 Wurzhurg, Federal Republic of Germany Abbreviation. 2',5'-ADP--epharose, adenosine 2',5'-bisphosphate liiiked to Sepharose via a 6-aminohcxyl group. Enzymes. D-Glucose-h-phosphate :NADP' oxidoreductase (EC 1.1.1.49); 1.-1actate:NAD' oxidoreductase (EC 1.1.1.27); urea amidohydrolase (EC 3.5.13;L-glutamatc: NAD(P)+ oxidorcductasc (deaminating) (EC 1.4.1.3); D-fructose-I ,6-bisphosphale D-glyceraldehyde-3-phosphate-lyasc (EC 4.1.2.13); ATP:pyruvate 0'phosphotransferase (EC 2.7.1.40); 5,6-dihydropyrimidine: NADP+ oxidoreductase (EC 1.3.1.2); hydrogen peroxide: hydrogen peroxide oxidoreductase (EC 1 .I 1 .I .6); P-~-gaIactosidegalactohydrolase (EC 3.2.1.23); ribonuclease A (EC 3.1.27.5); 5,6-dihydroorotate:02 oxidoreductase (EC 1.3.3.1).

NADPH, NADP', FAD and FMN were purchased from Boehringcr Mannheim GmbH. Uracil, aminoethylisothiouronium bromide, phenylmethylsulfonyl fluoridc and dithioerythritol were obtained from Sigma. Benzamidine hydrochloride was a product of Aldrich Chemical Co. Sucrose and DEAE-cellulose were obtained from Merck, Darmstadt, while 2',5'-ADP- Sepharose, Sepharose 5-400 and Mino RP C2/C18-HPLC column were purchased from Pharmacia. PMlO and PM30 ultrafiltration mcmbranes were obtained from Amicon, Inc., and Chelex 100 from BioRad. All other chemicals and reagents were obtained from commercial sources and were of the highest purity available. Enzyme ussuy

Enzyme activity was determined at 37°C by monitoring the decrease in absorbance at 340 nm accompanying the conversion of NADPH to NADP'. A typical reaction mixture contained: 28 mM potassium phosphate pH 7.4, 2 m M MgC12, 0.8 mM dithioerythritol, 60 pM NADPH, 150 pM uracil and enzyme solution in a final volume of 1 ml. The reaction was initiated with uracil and run against a blank containing the identical reaction mixture without uracil. The enzyme activity was the difference between the rate observed for the full reaction mixture minus that of the blank. For kinetic experiments, appropriate amounts of products or substrate analogs were added to the test and blank cuvette and

220 the assay was carricd out as discussed above. One unit of enzyme activity was defined as the amount of enzyme that causes the disappearance of 1 pmol NADPH/h. Enzyme purification All steps of the purification were carried out at 4°C. Crude extract. Freshly excised pig liver (1 50 g) was minced and homogenized in 250 ml buffer A containing 35 mM potassium phosphate pH 7.4 and 2.5 mM MgC12,in the presence of 1 mM aminoethylisothiouronium bromide, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, 10 mM EDTA and 2 mM dithioerythritol. The slurry was centrifuged at 27 600 x g for 30 min followed by an additional centrifugation at 100000 x g for 60 min. Ammonium sulfate fractionation. Solid (NH4)2S04 was then slowly added to the supernatant of the second centrifugation to give a final concentration of 30% saturation. After 30 min of additional stirring, the mixture was centrifuged at 43 500 x g for 15 min. Further (NH4)2S04was added to the supernatant until a concentration of 55% saturation was reached. The solution was stirred for an additional 30min and the precipitated protein was collected by centrifugation. The 30 - 55% (NH4)2S04precipitate was dissolved in about SO ml buffer A containing 1 mM dithioerythritol and dialyzed two times against 1 1 buffer A plus 1 mM dithoerythritol. Treatment at p H 4.85. To the dialyzed ammonium sulfate fraction, 5 % acetic acid was added with stirring to give a final pH of 4.85. After further stirring for 10 min at this pH, the enzyme solution was centriluged for 10 min at 43500 x g. The supernatant was readjusted to pH 7.3 with 0.5 M NaOH and dialyzed twice against 1 1 buffer A plus 0.5mM dithioerythritol. DEAE-cellulose chroivmtography. After dialysis, the enzyme solution was centrifuged for 10 min at 43500 x g and applied to a DEAE-cellulose column (2.5 x 30 cm), equilibrated with buffer A plus 0.5 mM dithioerythritol. The column was washed with the same buffer until the 280-nm absorbance indicated no further protein was being eluted. A linear gradient (total volume 600 ml) of KC1 (0-0.25 M) in buffer A plus 0.5 inM dithioerythritol was used to elute the enzyme. The enzyme appcared in the 0.015-0.045 M KC1 region. The pooled activc fractions were concentrated to about 10 ml by ultrafiltration on a PM30 membrane (Amicon) in a stirred cell. 2’,5’-ADP - Sepharose affinity chromatography. After dialysis twice against 1 1 buffer A plus 0.5 mM dithioerythritol, the enzyme solution was applied to a 2’,5’-ADP - Sepharose column (1.5 x 11 cm), equilibrated with buffer A plus 0.5 mM dithioerythritol. The affinity column was washed with equilibration buffer until all unbound protein had been eluted, followed by a linear gradient (total volume 600 ml) of KC1 (0 - 2 M) in buffer A plus 0.5 mM dithioerythritol. Dihydropyrimidine dehydrogenase fractions (0.1-0.2 M KCI) were pooled, concentrated by ultrafiltration on a PM30 membrane (Amicon) and dialyzed against buffer A plus 0.5mM dithioerythritol. The purified dihydropyrimidine dehydrogenase has a specific activity ranging over 20 - 25 units/mg protein. Enzyme was stored at 4°C in buffer A plus 0.5mM dithioerythritol. Flavin determination The purified enzyme dissolved in 35 mM potassium phosphate pH 7.4, 2.5 mM MgC1, and 0.5 mM dithioerythritol

was boiled in a water bath for 10 min in the dark to liberate flavin. After removing the precipitate by centrifugation, aliquots of the supernatant were analyzed qualitatively for flavin composition by HPLC separation on a Mino RP C2/C,, column with a linear gradient (0-66% methanol) in 20 mM potassium phosphate pH 5.6, at a flow rate of 1 ml/min at 25 “C. Flavins were detected at 230 nm. The FADiFMN composition of the supernatant was analyzed quantitatively by fluorescence measurements at different pH values [9] with commercially available samples of FAD and FMN purified on DEAE-cellulose [lo] as standards. The dihydropyrimidine dehydrogenase used had a protein concentration of 0.305 mg/ mi. Metal, acid-lubilc. sulfide and protein determination Analysis of metal contcnt was carried out by atomic absorption spectroscopy on the purified enzyme using a PerkinElmer atomic absorption spectrophotometer model 3030 while the quantitative determination of acid-labile sulfide was accomplished using the methylene blue method [ll]. Protein was determined by precipitation and detection using amido black [12] or by the Lowry procedure [13]. Electrophoresis Native disc gel electrophoresis was performed on 8 -25% gradient gels with the Phast System (Phannacia) using bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), glucose-6-phosphate dehydrogenase (216 kDa), catalase (240 kDa) and glutamate dehydrogenase (320 kDa) as protein standards. SDS/PAGE was carried out as described by Weber et al. [14] using 28% polyacrylamide slab gels. Gels were stained for protein with silver [I51 or with Coomassie brilliant blue K in 10% acetic acid in methanol/H20 (1 :1, by vol.) and destained in 5% acetic acid, 7.5% methanol in H 2 0 (by vol.). Relative mobilities of protcin bands were calculated with respect to the following markers: fl-galactosidase (1 15 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). Isoelectric focussing was accomplished with the Phast System (Pharmacia) using standard proteins from Serva (pH range 3 - 9). Analytical gel Jiltration Gel filtration chromatography was carried out with a column (1.6 x 92.5 cm) of Sephacryl S-400 equilibrated with 50 mM sodium phosphate pH 7.0 containing 150 mM sodium chloride and 1 mM dithioerythritol at room temperature. Samples of 0.5 ml were applied to the column followed by additional 0.5 ml buffer containing 10%osucrose. Reference proteins [urease (480 kDa), pyruvate kinase (237 kDa) and aldolase (158 kDa)] were located by activity. Bovine serum albumin (67 kDa) was detected by 280-nm absorbance and SDSIPAGE. Amino acid content Dihydropyrimidine dehydrogenase was dialyzed exhaustively against water prior to amino acid analysis. After hydrolyzing the enzyme in 6 M HCI at 110°C for 16 h, amino acids were separated and analyzed on a Biotronic C 2000 amino acid analyzer. Cysteine and methionine residues of

22 1 Table 1. Purfirntion of dihydropyrimidine dehydrogriiase sturting with 150 gfreshly excisedpig liver Purification step

Crude extract Ammonium sulfate fractionation” pH 4.85 treatmentb DEAE-ccllulosc eluatc 2’,5’-ADP- Scpharose eluate a

Volume

Total protein

Total activity

Specific activity

Purification

Yield

ml

mg

units

units/mg

285 132 144 194 165

28 400 6100 5 700 200 2.6

193 191 188 110 55

0.0068 0.031 0.033 0.55 21

fold 1 4.6 4.9 81 3100

Yo 100 99 97 57 28

After dialysis. After dialysis and centrifugation

1

dihydropyrimidine dehydrogenase were oxidized by formic acid and hydrogen peroxide [16] prior to hydrolysis.

Amino acid sequence determination Amino-terminal sequences were determined by automated Edman degradation with a gas-phase sequencer (model 470A, Applied Biosystems Inc. Forster City, CA, USA). The purified dihydropyrimidine dehydrogcnase was first separated on 18% SDSIPAGE and the protein bands were transblotted onto a polyvinylidene difluoride membrane (Immobilon, Millipore). The blotted proteins were detected by staining with Coomassie brilliant blue R. The electroblotted polypeptides corresponding to the stained bands were cut out and degraded directly on the PVDF membrane by the Edman procedure. Phenylthiohydantoin derivatives of amino acids were analyzed by HPLC on an RP 18 column (Applied Biosystems) using a h e a r gradient of 17-38% solvent B in solvent A (solvent A = 50/0 tetrahydrofuran, 0,054 M sodium acetate pH 3.8,0.0117 M sodium acetate pH 4.6.0.0125% trimethylamine in H 2 0 ; solvent B: 500 nM dimethylphenylthiourea in acetonitrile). C y a n o g e n bromide cleavage

Proteins separated on 18% SDSjPAGE were stained with Coomassie brilliant blue R, bands were cut out and treated separately with cyanogen bromide for 16 h. The cleavage solution contained 35 mg/ml cyanogen bromide in formic acid/ H 2 0 (7 : 3 , by vol.). After removing the solvent, products were analyzed on 18% SDSIPAGE. Blanks were run in the presence of cleavage solution but without cyanogen bromide. RESULTS AND DISCUSSION A typical purification procedure for dihydropyrimidine dehydrogenase from pig liver is summarized in Table 1. Fig. 1 illustrates the SDSjPAGE pattern of individual purification steps. Dihydropyrimidine dehydrogenase was purified 3100fold with a specific activity of 20 - 30 unitsjmg protein and a 28% recovery. Purified enzyme at protein concentrations of 0.5 - 1 mg protein/ml was stable at - 20 “C in the presence of 1 mM dithioerythritol for several months without significant loss of enzymatic activity. The purified enzyme was homogeneous as judged by gel filtration on Sephacryl S-400 showing a single, symmetrical peak corresponding to a molecular mass of 206 kDa. This valuc is in good agreement with other reports on the rat liver enzyme [8, 171. A molecular mass of 204 kDa was obtained with the P h a s t System and commercially availabie 8 -25%

115 k D a 67 k D a -

2

3

4

5

6

7 -107 k D a - 92 k D a

L3 kDa-

2 5 kDa13.7 k D a -

- 12 k D a

Fig. 1. SDSIPACE of various purcfication steps of dihydropyrimidine ifehydrogenase. Lane 1. J-galactosidase (standard protein); lane 2, bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonucleasc A (standard proteins); lane 3, crude extract; lane 4, ammonium sulfate 30- 5 5 % precipitate; lane 5, pH-4.85 supernatant; lane 6, pooled fractions from DEAE-ccllulose chromatography; lane 7, poolcd fractions from 2’,5’-ADP - Sepharose chromatography

gradient gels (Fig. 2). Dihydropyrimidine dehydrogenase from pig liver reveals three different bands on SDS gels with molecular masses of 107 kDa, 92 kDa and 12 kDa, respectively (Fig. 2). The binding capacity of the 12-kDa band for Coomassie brilliant blue R is very low but this band can clearly be seen in silver-stained gels. The question whether the three polypeptide bands in SDS/ PAGE are real subunits of the native protein or products of proteolytic degradation has been examined by sequencing the N-terminus of the three polypeptides. The first ten aminoterminal residues of the 107-kDa and 92-kDa peptides were found to be identical (Table 2). Therefore it is very likely that the native dihydropyrimidine dehydrogenase consists of two subunits with equal molecular masses (107 kDa). The 12kDa peptide is formed by proteolytic attack at the carboxylterminal of the 107-kDa subunits leaving a 92-kDa fragment. This hypothesis is supported by experiments in which the three polypeptides are cleaved with cyanogen bromide. The cyanogen bromide SDS pattern of the 107-kDa and the 92kDa proteins are apparently identical (Fig. 3). Cyanogen bromide treatment of the 12-kDa peptide results in some peptides with a greater molecular mass than the original sample and

222 Table 2. N-terminal sequences of electroblotted pdypeptide chains of dihydropyrimidine dehyhgenuse from pig liver after separation on SDSf PAGE Chain

Residue 1

107 kDa 92 kDa 12 kDa

5

Val Val

Leu Pro

Phe

LYs LYs

Ser

Leu

Ser Pro

Le 11

10

ASP

Val Val Xaa

ASP

Glu

6.01

Ala Ala Xaa

ASP

ASP

Pro

1le I1 e

Glu Ile

Phe

E

5.5-

Table 3. Amino acid compositions ofdihydropyrinzidine dehydrogcnases jrom pig and rat liver [ 8 / n.d. = not determined

0

5.0~

:

Amino acid

4.5-

VI

VI L

3

Amount in enzyme from

0

E

pig liver mol/mol enzyme

- 4.03.54 0.0

rat liver

.

I

0.2

.

I

,

I

0.4 Mobility

0.6

,

T_-

0.8

Fig. 2. Molecular mass determination of' native dihydropyrirnidine deitydrogenase and the three polypeptide chains separated by SDSIPAGE. The first five proteins were used for native 8-25% gradient PAGE, whereas the latter five proteins were employed for SDSIPAGE. Protein standards were glutamate dehydrogenase (I), catalase (2), glucosc-6-phosphate dchydrogenase (3), lactate dchydrogenase (4), bovine serum albumin (9,j-galactosidasc (6), bovine serum albumin (7). ovalbumin (8), chymotrypsinogen A (Y) and ribonuclease (10). The circle indicates the position of native dihydropyrimidine dehydrogenase, the squares indicate the position of the three SDS bands

1

2

3

4

5

6

107 kDa, 92 k D a -

CYS Met 4sp Thr Ser Glu Pro GlY Ala Val Ile Leu TYr Phe Lys His .4rg TrP

+ Asn + Gln

30.7". 94.0" 177.7 109.6 119.4 196.4 126.2 185.4 171.5 108.4 106.9 167.4 30.3 74.3 124.1 22.0 72.7 n.d.

27.9 41.8 185.1 102.7* 121.9d 141.2 113.3 121.1 121.9 86.2 99.9 134.1 35.9 64.1 92.0 22.3 63.0 n. d.

When cysteine was determined as cysteine sulfonic acid

Y9.2 mol/mol enzyme was found.

Determined by titration with 5,5'-dithiobis(2-nitrobcnzoate). ' Detcrmincd as methionine sulfoxide. Value extrapolated to zero time.

12 k D a -

Fig. 3. Cyanogen bromide,fragments of the three SDSIPAGEpolypeptide chains. Dihydropyrimidine dehydrogenase was first separated into three polypeptide chains by SDSjPAGE, the individual hands were cut out (107-kDa, 92-kDa and 12-kDa bands) and treated with cyanogen bromide (CNBr). Lane 1, CNBr fragments of the 107-kDa band; lane 3, CNBr fragments of the 92-kDa hand; lane 5, CNBr fragments of the 12-kDa band; lanes 2, 4, 6, separated chains had been treated in the same manner as in lanes 1, 3 and 5 in the absence of CNBr

some smaller fragments. Control experiments shown in lanes

2, 4 and 6 of Fig. 3 reveal undigested, original polypeptides.

The result of the cyanogen-bromide-treated 12-kDa peptide, together with its observed low affinity to Coomassie brilliant

blue R, indicates that this peptide is highly hydrophobic and tends to aggregate during cyanogen bromide treatment. We were unable to abolish the protease attack completely by using different protease inhibitors, such as phcnylmethylsulfonyl fluoride, z ,-antitrypsin, aprotinin, benzamidine, pepstatin A, leupeptin or metal chelators like EDTA or 1,lO-phenanthroline. Determination of the isoelectric point of dihydropyrimidine dehydrogenase from pig liver by isoelectric focusing resulted in a PI of approximately 4.65. Compared with the rat liver enzyme (PI 5.25) a larger number of acidic amino acids must be located on the surface of the pig liver enzyme. Table 3 compares the amino acid composition of the pig liver enzyme with the rat liver enzyme. The purified enzyme appears yellow in color and has an absorption spectrum with maxima at 269 nm, 375 nm and 432nm and a shoulder at 328 nm (Fig. 4A). indicating an atypical flavoprotein. The absorption ratios were A269:A32S:A375:A423 = 3.0:1.0:1.1:1. The ratio of A 2 7 3: A454 is about 3.7 for dihydroorotate dehydrogenase from Zymobacterium oroticum [IS].

223 0.6 o)

u

+I o

n Q

0.5 0.4

0.3

A

4i 269

Table 4. Iron, acid-labile sulfide andflavin content of dihydropyrimidine dehy dr ogenase Substance assayed

I\

Amountjassay of protein

substance

0.2

82.9 155.0

0.76 1.22

mol/mol enzyme 32.8 28.0

Inorganic sulfur

16.4 65.5

0.08 0.36

28.8 33.8

FMN

52.4 65.5

0.20 0.25

1.7 1.7

FAD

52.4 65.5

0.32 0.49

1.6 1.9

0.1

0.0

250

Iron 300

350

400

450

500

550

600

Wavelength [nm]

B

"'""T

Wavelength [nrn]

f i g . 4. Absorption spectra of' purified dihydropyrimidine dehydroge-

nase. (A) Purified enzyme (-j and FMN (- - -) in buffer A plus 1 mM dithioerythritol at concentrations of 0.284 mg/ml and 13.2 pM, respectively. (B) Difference spectra of the dehydrogenase (0.335 mgj ml) in buffcr A plus 1 mM dithioerythritol in the presence of various

concentrations of uracil. The reference cuvette contained enzyme and buffer A plus 1 mM dithioerythritol in the appropriate volume. Final concentrations of uracil wcre 9.6 pM, 18.7 pM, 82.0 pM and 200.0 pM, rcspectively. The spectra obtained with increasing uracil concentrations are shown from bottom to top; they are uncorrected for dilution

The nature of the flavin cofactor was determined after its dissociation from the protein. Thus, purified enzyme (in buffer A) was boiled in a water bath for 10 min in the dark. After removing precipitated protein by centrifugation, the flavins were separated by HPLC. Standard solutions of FAD and FMN were treated in the same way as enzyme. No conversion of FAD to FMN could be detected under those experimental conditions. FAD and FMN were analyzed quantitatively by a simultaneous fluorometric assay. As documented in Table 4, dihydropyrimidine dehydrogenase contains nearly two moles each of FAD and FMN per mole of enzyme. Equal amounts of FMN and FAD were also found for dihydroorotate dehydrogenase [18]. In contrast 4 rnol FAD were found in 1 mol rat liver enzyme [8]. The low absorption ratio (,4269/A432= 3), suggests a complex flavoprotein with additional absorbance in the visible region which might be due to iron-sulfur centers. For analysis of metals known to be involved in the oxidative-reductive processes, e.g. iron, copper or zinc, the purified enzyme was subjected to atomic absorption spectroscopy. Nearly 32 mol iron/mol enzyme were found even after passing the purified dihydropyrimidine dehydrogenase through Chelex 100. Controls of buffer and water did not contain detectable amounts of iron. Shiotani and Weber [8] detected only 3 mol iron/ rnol in the rat liver enzyme which might be a result of their purification procedure (pH-4.85 precipitation prcceeding ammonium sulfate fractionation. no dialysis between thcse steps). Other metal ions were not present in pig liver

Amount in the enzyme

dihydropyrimidine dehydrogenase. The acid-labile sulfide content of the purified pig liver enzyme was analysed to determine the binding mode of the iron atoms. As illustrated in Table 4, the acid-labile sulfide content is equal to the iron content indicating the probable existence of x[Fe-S] centers. In buffer controls no sulfide was found. Considering the flavin content, there were 8 rnol each of iron and acid-labile sulfide/ mol flavine nucleotide. The redox state of the dehydrogenase in our preparations was determined by monitoring difference spectra in the presence and absence of substrate. Upon addition of uracil, absorbance between 350 - 500 nm is noticeably increased (Fig. 4B), suggesting that thc chromophores are partially reduced. These spectral changes appeared to saturate at 200 pM uracil. Dihydropyrimidine dehydrogcnasc exhibits its maximum enzymatic activity in the pH range 7.2-7.5. When the pH value is decreased to 6.0, enzymatic activity is reduced to 50% of its maximum value, probably as a result of the destruction of NADPH in an acid environment. At pH 8.0, enzymatic activity again is reduced to 50% with drastic further reduction at higher pH values. Natural substrates of dihydropyrimidine dehydrogenase are thymine and uracil, where uracil is reduced at a reaction velocity of 129% that of thymine. Substrate analogues that have been found to be converted by dihydropyrimidine dehydrogenase are uracil derivatives substituted in the 5-position like 5-aminouracil and 5-fluorouracil, which exhibit reaction velocities of 146% and 156% that of thymine, respectively. When the reaction velocities in Table 5 are inspected it seems obvious that an electron-withdrawing substituent (H, NH2, F) on carbon 5 of the pyrimidine substrate is essential for the rate of the enzymatic reduction. The apparent lunetic parameters of these dihydropyrimidine dehydrogenase substrates have been determined. With standard assay conditions at 3 7 ° C the amount of reduction was proportional to the incubation time with 1- 5 pg enzyme protein for up to 5 min. At 60 pM NADPH, substrate inhibition was obtained for all pyrimidine substrates examined. At varying uracil concentrations the Lineweaver-Burk plot is linear up to 20 pM uracil (Fig. 5A). From the linear portion on the double-reciprocal plots apparent K, values for thymine, uracil, 5-aminouracil and 5-fluorouracil were obtained and they are compiled in Table 5. Thc steady-state affinities for thymine and uracil are in the same range with apparent K, values of 2.66 and

224 Table 5. Comparison of kinetic parameters und substrate .rpecijiiities of dihydropyrimidine dehydrogenase Apparent K,,, values were calculated using the non-lincar regression data analysis program Enzfitter (Elsevier Science Publisher BV). Assays were performed using the standard conditions described in MaLerials and Methods. The specific activity was measured at 100 pM substrate concentration and 60 pM NADPH. Relative activity was calculatcd taking the specific activity when thymine was used as substrate as 100%. The K, of NADPH was mcasured at 100 pM uracil Substrate

Thymine Uracil 5-Aminouracil 5-Fluorouracil NADPH

Apparent K,

Specific activity

Relativc activity

UM

U/mg

?b

15.22 19.56 22.21 23.78

100 128.6 146 156.2

2.66 1.w

5.10 5.50 11.36

I/V

A

and C-6 occurs. The turnover of uracil reduction is severely inhibited by 2,6-dihydroxypyridine. At 100 pM uracil we observed substrate inhibition by NADPH (Fg. 5B). From the linear portion of the doublereciprocal plot ranging up to 20 pM NADPH an apparent K, value for NADPH of 11.4 pM can be calculated. The kinetic data are summarized in Table 5. The K, valucs for uracil, thymine and NADPH were found to be in the same range as those reported for the rat liver enzyme except that for the dehydrogenase from rat liver no substrate inhibition is reported [S]. Substrate inhibition, however, was found for dihydropyrimidine dehydrogenase in crude extracts of some normal and neoplastic human tissues [19]. In conclusion, we believe that dihydropyrimidine dehydrogenase from pig liver is a complex iron-sulfur flavoprotein. Further purification and characterization of the three polypeptide chains in SDS gels, examination of the proposed FeS clusters and their role in the catalytic process as well as production of antibodies against the pig liver enzyme are currently in progress. Wc arc greatly indebted to Professor Paul F. Cook (Texas College of Osteopathic Medicine, Fort Worth, Tcxas, USA) for valuable advice and his great help in revising the manuscript. We are also indebted to Profcssor Jurgcn Hoppc and to Dr Vivian Hoppc for the amino acid analysis and sequence studies. We thank Mr Rommelt (Zentrallabor Medizinische Klinik Wiirzburg) for the metal determinations and Mr Gunther Waldmann and Mr Jurgen Kautz for stimulating discussions. This study was supported in part by the Ueutsche Forschungsgemeinschaft (Schn 139/9-2).

[u/rn1]-1

1.0

REFERENCES -0.50

-0.25

0.00

-0.2

-0.1

0.0

,

0.25

.

,

0.1 I/NADPH

0.50

0.2

[PM-~]

Fig. 5. Double-reciprocal plot of reaction velocity versus the concentration of ( A ) uracil and ( B ) N A D P H . Conditions: 35 mM potassium phosphate pH 7.3,2.5 mM MgC12, 1 mM dithoerythritol in the presence of60 pM NADPH (A) and 100 pM uracil (B)

1.98 pM, rcspcctively. The apparent K, values for 5aminouracil and 5-fluorouracil are twice as high (Table 5). Dihydropyrimidinc dehydrogenase does not use cytosine or orotate as substrate. The free 0 x 0 o r hydroxyl group at carbon 4 seems to be essential in the dehydrogenase rcaction. If this group is substituted by an amino function (cytosine), no reduction of the pyrimidine double bond between C-5

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