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[55] Bacterial folypoly(γ-glutamate) synthase-dihydrofolate synthase
349
FOLYLPOLYGLUTAMATESYNTHASE
KLSI
I
I
I
I
IO
20
30
40
so
nM unlabeled PteGlu
FIG. 2. The competitive inhibition of the reduction of [‘H]PteGlu to [)H]H,PteGlu by unlabeled PteGlu at pH 7.2.
Comments
There are a number of advantages of this radioenzymatic assay. First, this method measures the activity of the enzyme using HzPteGlu, the more physiological substrate. Second, the coupling of the chemical reduction of [3H]PteGlu to [3H]H$‘teGlu with the enzymatic reaction eliminates the need for the complicated synthesis and storage of the labile [3H]H#teGlu. Third, depending on the specific activity of [3H]PteGlu, the assay is very sensitive and can monitor the reduction of as little as 0.5 nM of substrate. Finally, this radioenzymatic assay for DHFR measures directly the reduction of the substrate rather than the oxidation of NADPH, so that enzyme activity can be determined in crude tissue or cell preparations which may contain other components which can oxidize NADPH.
FolylpolyCy-glutamate) Synthase-Dihydrofolate Synthase
[%I Bacterial
By ANDREW L. BOGNAR and BARRY SHANE
Folylpolyglutamates, the major cellular forms of the vitamin, are the active coenzymes for the reactions of one-carbon metabolism. Folypolyglutamate synthase (EC 6.3.2.17), the enzyme that catalyzes the conversion of pteroylmonoglutamates to poly (-y-glutamate) derivatives, has METHODS IN ENZYMOLOGY,
VOL. 122
Copyright B 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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been purified to homogeneity from Corynebacterium,’ Lactobacillus casei,= and Escherichia colk3 Protein purified from bacteria that biosynthesize folate de nouo also possessesdihydrofolate synthase activity.ls4 The Corynebacterium protein catalyzes the following reactions: Dihydropteroate + MgATP + r&ttamate + dihydrofolate + MgADP + Pi Tetrahydrofolate + MgATP + L-glutamate + tetrahydropteroyldiglutamate + MgADP + Pi 5,10-Methylenetetrahydropteroyl(glutarnate)~ + MgATP + L-glutamate + 5,10-methylenetetrahydropteroyl(glutamate)~+~ + MgADP + Pi
where n equals 1 to 3. Protein purified from Lactobacillus, an organism that requires exogenous folate for growth, lacks dihydrofolate synthase activity, and catalyzes the following reaction: 5,10-Methylenetetrahydropteroyl(glutamate), + MgATP + rglutamate + 5,10-methylenetetrahydropteroyl(glutamate),,+~
+ MgADP + Pi
where n can be from 1 to 10 in Go. In this report, the purification and general properties of the Corynebacterium and L. casei folylpolyglutamate synthases are described. Folylpolyglutamate Synthetase Assay Enzyme activity is normally measured by the incorporation of [i4C]glutamate into folylpolyglutamates using unlabeled (6R,S)-tetrahydrofolate and (6&Y)-5, lo-methylenetetrahydrofolate as the folate substrates for the Corynebacterium and Lactobacillus enzymes, respectively. ‘y2s5 Reaction mixtures contain 100 mM Tris-50 miV glycine buffer (pH 9.75 at 22% (6&S)-tetrahydrofolate (100 PM), formaldehyde (5 mM; Lactobacillus enzyme only), L-[*4C]glutamate (250 pM; 1.25 &i), ATP (5 mM), MgC12(10 mM), KC1 (200 mJ4), dithiothreitol(5 mA4), 2-mercaptoethanol (10 n&f; derived from folate solution), dimethyl sulfoxide (50 pl), bovine serum albumin (50 pg), and enzyme in a total volume of 0.5 ml. The reaction tubes are capped and incubated at 37”for 2 hr. The reaction is stopped by the addition of ice-cold 30 m/t4 P-mercaptoethanol (1.5 ml) containing 10 miV glutamate, and the mixture is applied I B. Shane, J. Biol. Chern. 255, 5655 (1980). 2 A. L. Bognar and B. Shane, J. Biol. Chem. 258, 12574 (1983). ’ A. L. Bognar, C. Osborne, B. Shane, S. C. Singer, and R. Ferone, J. Biol. Chem. 260, 5625 (1985). ’ R. Ferone and A. Warskow, Adv. Exp. Med. Biol. 163, 167 (1983). 5 J. J. McGuire, P. Hsieh, J. K. Coward, and J. R. Bertino, J. Biol. Chem. 255,5776 (1980).
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to a DEAE-cellulose (DE-52, Whatman) column (2 X 0.7 cm), protected by a 3-mm layer of nonionic cellulose, that has been equilibrated with 10 mM Tris buffer (pH 7.5) containing 80 mkf NaCl and 30 mM mercaptoethanol. Unreacted glutamate is eluted with the equilibration buffer (3 x 5 ml) and the labeled folate product is eluted with 0.1 N HCl (3 ml). Dihydrofolate Synthase Assay The dihydrofolate synthase assay is identical to the folylpolyglutamate synthase assay except dihydropteroate (50 @) replaces the folate substrate . Growth of Organisms Corynebacterium is cultured in medium containing (per liter) glucose (20 g), glycine (4 g), K2HP04 * 3H20 (0.66 g), KH#O, (0.5 g), MgS04. 7H20 (200 mg), FeS04. 7H20 (40 mg), and thiamine. HCl (1 mg). The organism is grown at 30“with vigorous aeration for 2-4 days (AM ,,,,,- 15). The pH is maintained between 6.7 and 7.0. Addition of low levels of yeast extract improves the growth rate without significantly affecting the yield of enzyme. Lactobacillus casei (ATCC 7469) is cultured in a complex medium containing 2 ti folic acid.6 Bacteria are grown at 37”without aeration for 22 hr. The pH of the medium drops from 6.5 to 4.8. Increased cell yield can be obtained by controlling the pH at 6.1, but the specific activity of the enzyme is reduced. Bacteria are collected by centrifugation, washed several times with ice-cold 0.9% NaCl, and resuspended in 1 volume ice-cold 50 mM potassium phosphate buffer (pH 7), containing 50 mM KC1 and the suspension is rapidly frozen as pellets and is stored at -80” until used. Purification of the Corpebacterium
Enzyme’
A typical purification of the Corynebacterium folylpolyglutamate synthase is shown in Table I. The purification obtained is about 7000-fold, and the purity of the final preparation ranges from 95 to lOO%, as judged by sodium dodecyl sulfate-gel electrophoresis. Dihydrofolate synthase activity copurifies with folylpolyglutamate synthase at a constant ratio of specific activities. 6 T. Tamura, Y. S. Shin, M. A. Williams, and E. L. R. Stokstad, Anal. Biochem. 49, 517 (1972).
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TABLE I PUR~FICATIONOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE SYNTHASE FROM Corynebacterium
Volume (ml)
Fraction 1. 2. 3. 4. 5. 6.
Crude extract O-50% ammonium sulfate DEAE-cellulose Sephadex G-150 Butyl-agarose AMP-agarose 0 Folylpolyglutamate
420 150 10 12 10 11
Protein (mid 4100 3210 17.5 1.78 0.29 0.088
Specific activity” (pmoI/hr . mg)
Purification (-fold)
Yield (%I
0.0049 0.0058 0.846 5.76 24.4 34.3
1.0 1.2 173 1176 4970 6994
100 94 74 51 35 15
synthase activity.
Step 1. Crude Extract. All operations are carried out at O-4”. The bacterial suspension is thawed, diluted with 1 volume of 50 mM potassium phosphate buffer (pH 7) containing 50 m&f KCl, and sonicated for 9 min using a Branson W-350 sonicator in the pulsed mode (30 min at 0.3 s/s). The sonicate is centrifuged and the supernatant dialyzed overnight against 50 mM potassium phosphate buffer (pH 7) containing 50 mI14KC1 to give a crude enzyme extract. The enzyme is unstable in the absence of KCl. Step 2. Ammonium Sulfate Fractionation. Ammonium sulfate is slowly added to the crude extract to give a 50% saturated solution. The pH should be readjusted to 7, if necessary. After stirring for 1 hr, the suspension is centrifuged and the precipitate is redissolved in 50 m&I potassium phosphate buffer (pH 7) containing 50 mM KC1 and dialyzed overnight against two changes of the same buffer. A 20-50% ammonium sulfate cut gives similar results. Step 3. DEAE-Cellulose Chromatography. Fraction 2 enzyme is applied to a DEAE-cellulose (DE-52; Whatman) column (40 x 2.5 cm) that has been equilibrated with 50 m&I potassium phosphate buffer (pH 7) containing 50 mM KCl. The column is washed with the equilibration buffer (200 ml) and eluted with a linear gradient (2 liters) of KC1 (50-600 mh4) in 50 mMpotassium phosphate buffer (pH 7). The enzyme elutes at a higher KC1 concentration (-430 mM) than the bulk of the applied protein. Active fractions are combined and concentrated by ultrafiltration (PM10 membrane; Amicon) to a volume of -5 ml. The extract is diluted with 50 n&potassium phosphate buffer (pH 7) to an approximate KC1 concentration of 150 nnI4 and reconcentrated by ultrafiltration to a volume of 5 ml. Step 4. Sephadex Chromatography. Fraction 3 enzyme is applied to a Sephadex G-150 column (90 X 2.6 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7) containing 150 mM KCl. The column is
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eluted with equilibration buffer and active fractions are combined and concentrated to -8 ml by ultrafiltration. Step 5. Butyl-Agarose Chromatography. Fraction 4 is dialyzed for 4 hr against 50 mM potassium phosphate buffer, pH 7 (200 volumes), and applied to a butyl-agarose (Miles Biochemicals) column (10 x 1 cm) equilibrated with the same buffer. The column is washed with the equilibration buffer (20 ml) and eluted with a linear gradient (100 ml) of KC1 (O-400 mh4) in buffer. The enzyme elutes after the majority of the protein from this column. Active fractions are combined and concentrated by ultrafiltration to about 5 ml. Step 6. AMP AfJinity Chromatography. Fraction 5 enzyme is dialyzed for 2 hr against two changes of 5 mM potassium phosphate buffer, (pH 7) containing 5 mM MgC& (100 volumes). Caution: the enzyme is unstable under these conditions. The dialyzed extract is applied to an AMP-hexylagarose (coupled through N6 of AMP; P-L Biochemicals) column (3 X 0.7 cm) equilibrated with the dialysis buffer. The column is washed with buffer (10 ml), buffer plus 2 mM ATP (5 ml), and buffer (10 ml), and purified enzyme is eluted with buffer plus 100 mM KC1 (10 ml). Fractions (5 ml) are collected in tubes containing 500 mM potassium phosphate buffer (pH 7) plus 500 mM KC1 (0.5 ml). Additional nonspecific protein is eluted from the column by buffer plus 500 mM KCl. The 60% loss of activity observed in this final purification step is due to the marked lability of the enzyme in the presence of low K+ and phosphate concentrations. Additional studies indicate that most of this loss in activity can be prevented by the inclusion of 10 or 30% dimethyl sulfoxide (Me*SO) in the wash and elution buffers. Me2S0 is added to Fraction 6 enzyme to a final concentration of 30% and the extract is divided into 0.5-ml aliquots and stored at -20, -80, or - 196”. Enzyme purified up to Step 4 is fully stable for at least 1 year when stored in 50 mM potassium phosphate buffer (pH 7) containing 150 mM KC1 at -20”. The purified preparation is unstable under these conditions and over 90% of the activity is lost in 1 week at -20” when stored in 50 mM buffer (pH 7) containing 50 mM KCl. Addition of Me$O (30%) stabilizes the preparation and no loss of activity is observed in 6 months when the preparation is stored at -20 to - 196”. The instability observed may be due to binding of the dilute protein preparation to glass or plastic containers.
Purification
of the Lactobacillus
Enzyme2
A typical purification of the L. casei enzyme is shown in Table II. The purification obtained ranges from 40,000- to 200,000-fold, depending on
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TABLE II PURIFICATION OF FOLYLPOLYGLUTAMATE SYNTHASE FROM Lactobacillus casei
Fraction 1. Crude extract 2. DEAE-cellulose 3. 30-60% ammonium sulfate 4. Phosphocellulose P-l 1 5. Phenyl-agarose 6. Sephadex G-100 7. AMP-agarose 8. lO-Aminodecyl-Sepharose
Volume (ml)
Protein (mg)
Specific activity (pmol/hr * mg)
5100 5700
38760 23655
0.00007 0.00013
1.0 1.9
0.00018 0.012 0.23 0.82 2.9 13.8
2.6 170 3300 11700 41000 197000
600 400 65 14 17 8.5
14400 330 9.75 1.36 0.17 0.014
Purification (-fold)
Yield (%I 100 106 87 81 77 38” 17 6.7
0 A higher yield of enzyme (74%) was recovered from the column but only part of this was used in the subsequent steps.
the growth conditions of the bacterial culture, and the purified preparation is homogeneous as judged by sodium dodecyl sulfate-gel electrophoresis. Step I. Crude Extract. All operations are carried out at O-4”. The bacterial suspension is thawed, diluted with 1 volume of 50 miI4 potassium phosphate buffer (PH 7) containing 50 mM KCl, and sonicated for 9 min using a Branson W-350 sonicator in the pulsed mode (30 min at 0.3 s/s). The sonicate is centrifuged and the precipitate resuspended in an equal volume of buffer and resonicated. This process is repeated two more times and the four supernatants obtained are combined to give a crude enzyme extract (Fraction 1). Step 2. DEAE-Cellulose Chromatography. Fraction 1 is applied to a DEAE-cellulose (DE-52, Whatman) column (40 x 7.5 cm) that has been equilibrated with 50 n&I potassium phosphate buffer (pH 7) containing 50 mM KCI. The column is washed with the equilibration buffer (700 ml) and the total flow-through is combined to give Fraction 2. This step is used primarily to remove nucleic acids and polynucleotides, but can result in up to a IO-fold purification when lower amounts of protein are applied to the column. Step 3. Ammonium Sulfate Fractionation. Ammonium sulfate is slowly added to Fraction 2 to give a 30% saturated solution. After stirring for 2 hr, the mixture is centrifuged and the precipitate discarded, Ammonium sulfate is slowly added to the supematant to give a 60% saturated solution and, after stirring for 2 hr, the mixture is centrifuged. The precipitate is resuspended in 50 mM potassium phosphate buffer, pH 7 (500 ml), and dialyzed overnight against the same buffer (40 volumes).
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Step 4. Phosphocellulose Chromatography. The dialyzed extract (Fraction 3) is applied to a phosphocellulose (P- 11, Whatman) column (11.3 x 7.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The column is washed with the equilibration buffer (800 ml) and eluted with a linear gradient (2 liters) of KC1 (O-250 mM) in the same buffer. Fractions containing the bulk of the enzyme activity are pooled (Fraction 4). Most of the applied protein does not bind to the phosphocellulose column. Step 5. Phenyl-Agarose Chromatography. Ammonium sulfate is added to Fraction 4 to give a 70% saturated solution and the mixture is stirred for 90 min and centrifuged. The precipitate is redissolved in 50 mM potassium phosphate buffer (pH 7.5), containing 0.41 it4 ammonium sulfate (10% saturation; pH after addition of ammonium sulfate) (23 ml) and the solution is applied to a phenyl-agarose (BRL) column (9 x 1 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.5), containing 0.41 M ammonium sulfate. The column is washed with buffer/O.41 M ammonium sulfate (30 ml) and buffer/O.33 M ammonium sulfate (30 ml) and eluted with a linear gradient (100 ml) of ammonium sulfate (0.33-o M) in buffer followed by buffer alone. Fractions containing enzyme activity, which elutes in the latter half of the gradient, are pooled. Step 6. Sephadex Chromatography. Fraction 5 enzyme is precipitated with ammonium sulfate (70% saturation) as described above, resuspended in 50 mM potassium phosphate buffer (pH 7), and applied to a Sephadex G-100 column (I 15 x 1.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The column is eluted with the equilibration buffer and fractions containing the majority of the enzyme activity are pooled and dialyzed for 3 hr against two changes of 5 mM potassium phosphate buffer, pH 7 (Fraction 6). Step 7. AMP Affinity Chromatography. Fraction 6 enzyme is applied to an AMP-hexyl-agarose (coupled through N6 of AMP; P-L Biochemicals) column (7 X 1 cm) equilibrated with 5 mM potassium phosphate buffer (pH 7), containing MgC12 (2 m&I). The column is washed with the equilibration buffer (20 ml) and eluted with 50 mM potassium phosphate buffer, pH 7 (15 ml) and 50 mM potassium phosphate buffer (pH 7) containing 250 mM KC1 (15 ml). The fractions with peak activity are pooled and dialyzed for 3 hr against two changes of 50 mM potassium phosphate buffer, pH 7 (Fraction 7). The enzyme is not eluted from this column by MgATP2-, suggesting that binding is due to an ion-exchange or phosphate affinity effect. Under the described conditions, most of the applied protein binds to the column and the enzyme is eluted before the bulk of the protein. Additional studies indicate that enzyme stability and recovery from this column are increased if the enzyme is eluted with 50 mM potassium phosphate buffer (pH 7) containing 10% Me2S0.
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TABLE III PROPERTIESOFBACTERIAL FOLYLPOLYGLUTAMATE SYNTHASW Cotynebacterium
Purification (-fold) pH optimum Monovalent cation Divalent cation Reducing agent M, SDS-gel electrophoresis (Sephadex) Dihydrofolate synthase activity Monoglutamate substrate Polyglutamate substrate Stabilizers
Lactobacillus
7000 9.5 200 mM K+ Mg2+ None
40,000-200,000 9.5 200 mM K+ Mg2+, Mn2+ None
53,000 51,000 Yes
43,000 43,000 No
H$teGlu 5,10-Methylene-H,PteGlu K+, Pi> Me2S0
5, IO-Methylene-H4PteG1u 5,10-Methylene-HrPteGlu K+, P, , Me,SO
a PteGlu, folic acid, pteroylglutamate; H,PteGlu, tetrahydropteroylpoly(-y-glutamate).
Step 8. Aminodecyl-Sepharose Chromatography. Fraction 7 is applied to a IO-aminodecyl-Sepharose column (7 x 1 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The bulk of the protein is eluted with the equilibration buffer (30 ml) and the enzyme is specifically eluted with buffer containing 250 mM KCl. Fractions containing enzyme activity are pooled. The purified protein is unstable, presumably due to the low protein concentration. MezSO is added to Fraction 8 enzyme to a final concentration of 20% (v/v) and the extract is divided into l-ml aliquots and stored at -20”. Approximately 50% of the enzyme activity remains after storage for 3 months. Properties
of the Purified
Proteins1*2,7,8
General properties of the purified proteins are listed in Table III. Both proteins are monomeric and have an absolute requirement for a monovalent cation for activity. K+ (200 mM) gives maximal stimulation of activity while Rb+ and NH4+ are active but less effective. The monovalent cation may cause a conformational change in the protein.9 The Mg2+ ’ B. Shane, in “Peptide Antibiotics-Biosynthesis and Functions” (H. Kleinkauf and H. Dohren, eds.), p. 353. de Gruyter, Berlin, 1982. * B. Shane and D. J Cichowicz, Adu. Exp. Med. Biol. 163, 149 (1983). 9 B. Shane, in “Chemistry and Biology of Pteridines” (J. A. Blair, ed.), p. 621. de Gruyter, Berlin, 1983.
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TABLE IV FOLATESUBSTRATESOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE
SYNTHASE
Relative activity (50 PM substrate) Values of n in Glu-,a
(6s IH&e
HJ’teGlu
(6R)-5,10-MethyleneH,PteGlu
Corynebacterium
(6R)-lo-FormylH,PteGlu
enzyme
20 100
1.5 0 Lactobacillus
46.3 33.3 0.2
11.5 0.4 0
casei enzyme
0.1 1.3 53 6.5
0.1 0.1 0 0
100
0.7
246 79 2.8 3.1 2.8 1.7
7.8 0.4
0.1 0 0 0
0 Subscript to Glu, n, denotes glutamate chain length in H,PteGlu,.
requirement is to generate the MgATP substrate and free ATP is a potent inhibitor of the reaction. The high pH optimum reflects the pK of the amino group of the glutamate substrate. The free amine form is the substrate for the reaction, and the reaction proceeds efficiently at pH 8 provided high levels of glutamate are provided. One mole of ATP is hydrolyzed to ADP and Pi for every mole of glutamate added to the folate or pteroate substrates. The folate substrate specificities of the enzymes are shown in Table IV. The Corynebacterium protein will use dihydropteroate and tetrahydrofolate as substrates. However, 5,10-methylene derivatives are the preferred polyglutamate substrates. The purified protein will metabolize labeled 5, lo-methylenetetrahydrofolate to the tetraglutamate derivative, which is the predominant polyglutamate found in viuo. The Lactobacillus enzyme will not utilize dihydropteroate as a substrate and preferentially uses 5,10-methylenetetrahydrofolate mono- and polyglutamate derivatives as its substrates. The purified L. casei enzyme will metabolize labeled 5, lo-methylenetetrahydrofolate primarily to the tetra- and pentaglutamate derivatives and small amounts of longer glutamate chain length derivatives are formed. The major in uiuo folates in Lactobacillus are octa- and nonaglutamate derivatives.
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TABLE V CONSTANTSOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE~YNTHASE
Wd Compound
(pmol/hr . mg) Cotynebacterium folylpolyglutamate synthetase
MgATP (6&Y)-H$teGlu L-Glutamate Phosphate p, y-Methylene-ATP ATP-y-S
18.0 2.1 160 -
10.0 12.7
MgATP HJ%eGlu L-Glutamate 8, y-Methylene-ATP ATP-y-S
Corynebacterium dihydrofolate synthetaseb 2.9 co.4 1380 1.0 0.29
4 750 0.58 3.0
Lactobacillus casei folylpolyglutamate synthetase MgATP 5600 800 2.3 15 (6R)-5,10-Methylene-H,PteGlu2 L-Glutamate 423 Phosphate 12,500 & y-Methylene-ATP 597 ATP-y-S 105
45.1 45.4 41.2
-
-
64.9 67.5 62.3 -
-
0 Not estimated or not applicable b Approximate values; detailed kinetic analyses not carried out.
The kinetic mechanism for both folylpolyglutamate synthases is ordered ter ter with the nucleotide substrate binding first, followed by the folate and glutamate substrates, and the order of product release is ADP, folate product, and Pi. z*OThis mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. A catalytic mechanism involving the formation of a folyl-y-glutamylphosphate intermediate followed by a nucleophilic attack by the free amine of glutamate on this mixed anhydride is suggested. Kinetic constants for the enzymes are shown in Table V. Dihydropteroate is a noncompetitive inhibitor of the Corynebacteriumfolylpolyghttamate synthaseactivity while tetrahydrofolate does not significantly inhibit the dihydrofolate synthase activity, suggestingthat the protein is bifunctional. The affinities of nucleotide analog inhibitors differ for the two I0 B. Shane, J. Biol. Chem. 255, 5663 (1980).
[551
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Corynebacterium synthase activities. As the nucleotide binds to free enzyme, this also suggests that the protein is bifunctional. Kinetic constants for the Lactobacillus folylpolyglutamate synthase are similar except for a greatly decreased afiinity for the nucleotide substrate, which is also reflected by a decreased affinity for nucleotide inhibitors. It should be noted that under the conditions of the standard assay, glutamate (250 PM) is not saturating for any of the activities and ATP (5 mM) is not saturating for the Lactobacillus enzyme. ATP is the preferred nucleotide substrate of both proteins although other purine triphosphates and UTP will substitute to some extent. ZTP is also a substrate for the Lactobacillus enzyme. The glutamate binding site is specific for L-glutamate although L-homocysteate and 4-fluoroglutamate are alternative substrates. Other Purified Folylpolyglutamate
Synthases
The E. coli folylpolyglutamate synthase-dihydrofolate synthase gene has recently been cloned and amplified and the protein has been purified to homogeneity.3 Its properties are similar to the Corynebacterium protein. It appears to be bifunctional and utilizes 5, lo-methylene derivatives as its polyglutamate substrate. The preferred monoglutamate substrate is lo-formyltetrahydrofolate.**J* The hog liver folylpolyglutamate synthase has been purified to homogeneity.13It has a slightly higher Kcatvalue than the bacterial enzymes. In contrast to the bacterial enzymes, the mammalian enzyme has an absolute requirement for a reducing agent, it can utilize oxidized folates as substrates, and the preferred polyglutamate substrates are tetrahydrofolate derivatives. Acknowledgment Supported in part by PHS Grant CA 22717 and Research Career Development Award CA 00697 (B.S.) from the National Cancer Institute, Department of Health and Human Services.
I’ M. Masurekar and G. M. Brown, Biochemistry 14, 2424 (1975). I* R. Ferone, S. C. Singer, M. H. Hanlon, and S. Roland, in “Chemistry and Biology of Pteridines” (J. A. Blair, ed.), p. 585. de Cruyter, Berlin, 1983. I3 D. J. Cichowicz and B. Shane, in preparation.
FOLYLPOLYGLUTAMATESYNTHASE
KLSI
I
I
I
I
IO
20
30
40
so
nM unlabeled PteGlu
FIG. 2. The competitive inhibition of the reduction of [‘H]PteGlu to [)H]H,PteGlu by unlabeled PteGlu at pH 7.2.
Comments
There are a number of advantages of this radioenzymatic assay. First, this method measures the activity of the enzyme using HzPteGlu, the more physiological substrate. Second, the coupling of the chemical reduction of [3H]PteGlu to [3H]H$‘teGlu with the enzymatic reaction eliminates the need for the complicated synthesis and storage of the labile [3H]H#teGlu. Third, depending on the specific activity of [3H]PteGlu, the assay is very sensitive and can monitor the reduction of as little as 0.5 nM of substrate. Finally, this radioenzymatic assay for DHFR measures directly the reduction of the substrate rather than the oxidation of NADPH, so that enzyme activity can be determined in crude tissue or cell preparations which may contain other components which can oxidize NADPH.
FolylpolyCy-glutamate) Synthase-Dihydrofolate Synthase
[%I Bacterial
By ANDREW L. BOGNAR and BARRY SHANE
Folylpolyglutamates, the major cellular forms of the vitamin, are the active coenzymes for the reactions of one-carbon metabolism. Folypolyglutamate synthase (EC 6.3.2.17), the enzyme that catalyzes the conversion of pteroylmonoglutamates to poly (-y-glutamate) derivatives, has METHODS IN ENZYMOLOGY,
VOL. 122
Copyright B 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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been purified to homogeneity from Corynebacterium,’ Lactobacillus casei,= and Escherichia colk3 Protein purified from bacteria that biosynthesize folate de nouo also possessesdihydrofolate synthase activity.ls4 The Corynebacterium protein catalyzes the following reactions: Dihydropteroate + MgATP + r&ttamate + dihydrofolate + MgADP + Pi Tetrahydrofolate + MgATP + L-glutamate + tetrahydropteroyldiglutamate + MgADP + Pi 5,10-Methylenetetrahydropteroyl(glutarnate)~ + MgATP + L-glutamate + 5,10-methylenetetrahydropteroyl(glutamate)~+~ + MgADP + Pi
where n equals 1 to 3. Protein purified from Lactobacillus, an organism that requires exogenous folate for growth, lacks dihydrofolate synthase activity, and catalyzes the following reaction: 5,10-Methylenetetrahydropteroyl(glutamate), + MgATP + rglutamate + 5,10-methylenetetrahydropteroyl(glutamate),,+~
+ MgADP + Pi
where n can be from 1 to 10 in Go. In this report, the purification and general properties of the Corynebacterium and L. casei folylpolyglutamate synthases are described. Folylpolyglutamate Synthetase Assay Enzyme activity is normally measured by the incorporation of [i4C]glutamate into folylpolyglutamates using unlabeled (6R,S)-tetrahydrofolate and (6&Y)-5, lo-methylenetetrahydrofolate as the folate substrates for the Corynebacterium and Lactobacillus enzymes, respectively. ‘y2s5 Reaction mixtures contain 100 mM Tris-50 miV glycine buffer (pH 9.75 at 22% (6&S)-tetrahydrofolate (100 PM), formaldehyde (5 mM; Lactobacillus enzyme only), L-[*4C]glutamate (250 pM; 1.25 &i), ATP (5 mM), MgC12(10 mM), KC1 (200 mJ4), dithiothreitol(5 mA4), 2-mercaptoethanol (10 n&f; derived from folate solution), dimethyl sulfoxide (50 pl), bovine serum albumin (50 pg), and enzyme in a total volume of 0.5 ml. The reaction tubes are capped and incubated at 37”for 2 hr. The reaction is stopped by the addition of ice-cold 30 m/t4 P-mercaptoethanol (1.5 ml) containing 10 miV glutamate, and the mixture is applied I B. Shane, J. Biol. Chern. 255, 5655 (1980). 2 A. L. Bognar and B. Shane, J. Biol. Chem. 258, 12574 (1983). ’ A. L. Bognar, C. Osborne, B. Shane, S. C. Singer, and R. Ferone, J. Biol. Chem. 260, 5625 (1985). ’ R. Ferone and A. Warskow, Adv. Exp. Med. Biol. 163, 167 (1983). 5 J. J. McGuire, P. Hsieh, J. K. Coward, and J. R. Bertino, J. Biol. Chem. 255,5776 (1980).
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SYNTHASE
3.51
to a DEAE-cellulose (DE-52, Whatman) column (2 X 0.7 cm), protected by a 3-mm layer of nonionic cellulose, that has been equilibrated with 10 mM Tris buffer (pH 7.5) containing 80 mkf NaCl and 30 mM mercaptoethanol. Unreacted glutamate is eluted with the equilibration buffer (3 x 5 ml) and the labeled folate product is eluted with 0.1 N HCl (3 ml). Dihydrofolate Synthase Assay The dihydrofolate synthase assay is identical to the folylpolyglutamate synthase assay except dihydropteroate (50 @) replaces the folate substrate . Growth of Organisms Corynebacterium is cultured in medium containing (per liter) glucose (20 g), glycine (4 g), K2HP04 * 3H20 (0.66 g), KH#O, (0.5 g), MgS04. 7H20 (200 mg), FeS04. 7H20 (40 mg), and thiamine. HCl (1 mg). The organism is grown at 30“with vigorous aeration for 2-4 days (AM ,,,,,- 15). The pH is maintained between 6.7 and 7.0. Addition of low levels of yeast extract improves the growth rate without significantly affecting the yield of enzyme. Lactobacillus casei (ATCC 7469) is cultured in a complex medium containing 2 ti folic acid.6 Bacteria are grown at 37”without aeration for 22 hr. The pH of the medium drops from 6.5 to 4.8. Increased cell yield can be obtained by controlling the pH at 6.1, but the specific activity of the enzyme is reduced. Bacteria are collected by centrifugation, washed several times with ice-cold 0.9% NaCl, and resuspended in 1 volume ice-cold 50 mM potassium phosphate buffer (pH 7), containing 50 mM KC1 and the suspension is rapidly frozen as pellets and is stored at -80” until used. Purification of the Corpebacterium
Enzyme’
A typical purification of the Corynebacterium folylpolyglutamate synthase is shown in Table I. The purification obtained is about 7000-fold, and the purity of the final preparation ranges from 95 to lOO%, as judged by sodium dodecyl sulfate-gel electrophoresis. Dihydrofolate synthase activity copurifies with folylpolyglutamate synthase at a constant ratio of specific activities. 6 T. Tamura, Y. S. Shin, M. A. Williams, and E. L. R. Stokstad, Anal. Biochem. 49, 517 (1972).
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TABLE I PUR~FICATIONOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE SYNTHASE FROM Corynebacterium
Volume (ml)
Fraction 1. 2. 3. 4. 5. 6.
Crude extract O-50% ammonium sulfate DEAE-cellulose Sephadex G-150 Butyl-agarose AMP-agarose 0 Folylpolyglutamate
420 150 10 12 10 11
Protein (mid 4100 3210 17.5 1.78 0.29 0.088
Specific activity” (pmoI/hr . mg)
Purification (-fold)
Yield (%I
0.0049 0.0058 0.846 5.76 24.4 34.3
1.0 1.2 173 1176 4970 6994
100 94 74 51 35 15
synthase activity.
Step 1. Crude Extract. All operations are carried out at O-4”. The bacterial suspension is thawed, diluted with 1 volume of 50 mM potassium phosphate buffer (pH 7) containing 50 m&f KCl, and sonicated for 9 min using a Branson W-350 sonicator in the pulsed mode (30 min at 0.3 s/s). The sonicate is centrifuged and the supernatant dialyzed overnight against 50 mM potassium phosphate buffer (pH 7) containing 50 mI14KC1 to give a crude enzyme extract. The enzyme is unstable in the absence of KCl. Step 2. Ammonium Sulfate Fractionation. Ammonium sulfate is slowly added to the crude extract to give a 50% saturated solution. The pH should be readjusted to 7, if necessary. After stirring for 1 hr, the suspension is centrifuged and the precipitate is redissolved in 50 m&I potassium phosphate buffer (pH 7) containing 50 mM KC1 and dialyzed overnight against two changes of the same buffer. A 20-50% ammonium sulfate cut gives similar results. Step 3. DEAE-Cellulose Chromatography. Fraction 2 enzyme is applied to a DEAE-cellulose (DE-52; Whatman) column (40 x 2.5 cm) that has been equilibrated with 50 m&I potassium phosphate buffer (pH 7) containing 50 mM KCl. The column is washed with the equilibration buffer (200 ml) and eluted with a linear gradient (2 liters) of KC1 (50-600 mh4) in 50 mMpotassium phosphate buffer (pH 7). The enzyme elutes at a higher KC1 concentration (-430 mM) than the bulk of the applied protein. Active fractions are combined and concentrated by ultrafiltration (PM10 membrane; Amicon) to a volume of -5 ml. The extract is diluted with 50 n&potassium phosphate buffer (pH 7) to an approximate KC1 concentration of 150 nnI4 and reconcentrated by ultrafiltration to a volume of 5 ml. Step 4. Sephadex Chromatography. Fraction 3 enzyme is applied to a Sephadex G-150 column (90 X 2.6 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7) containing 150 mM KCl. The column is
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eluted with equilibration buffer and active fractions are combined and concentrated to -8 ml by ultrafiltration. Step 5. Butyl-Agarose Chromatography. Fraction 4 is dialyzed for 4 hr against 50 mM potassium phosphate buffer, pH 7 (200 volumes), and applied to a butyl-agarose (Miles Biochemicals) column (10 x 1 cm) equilibrated with the same buffer. The column is washed with the equilibration buffer (20 ml) and eluted with a linear gradient (100 ml) of KC1 (O-400 mh4) in buffer. The enzyme elutes after the majority of the protein from this column. Active fractions are combined and concentrated by ultrafiltration to about 5 ml. Step 6. AMP AfJinity Chromatography. Fraction 5 enzyme is dialyzed for 2 hr against two changes of 5 mM potassium phosphate buffer, (pH 7) containing 5 mM MgC& (100 volumes). Caution: the enzyme is unstable under these conditions. The dialyzed extract is applied to an AMP-hexylagarose (coupled through N6 of AMP; P-L Biochemicals) column (3 X 0.7 cm) equilibrated with the dialysis buffer. The column is washed with buffer (10 ml), buffer plus 2 mM ATP (5 ml), and buffer (10 ml), and purified enzyme is eluted with buffer plus 100 mM KC1 (10 ml). Fractions (5 ml) are collected in tubes containing 500 mM potassium phosphate buffer (pH 7) plus 500 mM KC1 (0.5 ml). Additional nonspecific protein is eluted from the column by buffer plus 500 mM KCl. The 60% loss of activity observed in this final purification step is due to the marked lability of the enzyme in the presence of low K+ and phosphate concentrations. Additional studies indicate that most of this loss in activity can be prevented by the inclusion of 10 or 30% dimethyl sulfoxide (Me*SO) in the wash and elution buffers. Me2S0 is added to Fraction 6 enzyme to a final concentration of 30% and the extract is divided into 0.5-ml aliquots and stored at -20, -80, or - 196”. Enzyme purified up to Step 4 is fully stable for at least 1 year when stored in 50 mM potassium phosphate buffer (pH 7) containing 150 mM KC1 at -20”. The purified preparation is unstable under these conditions and over 90% of the activity is lost in 1 week at -20” when stored in 50 mM buffer (pH 7) containing 50 mM KCl. Addition of Me$O (30%) stabilizes the preparation and no loss of activity is observed in 6 months when the preparation is stored at -20 to - 196”. The instability observed may be due to binding of the dilute protein preparation to glass or plastic containers.
Purification
of the Lactobacillus
Enzyme2
A typical purification of the L. casei enzyme is shown in Table II. The purification obtained ranges from 40,000- to 200,000-fold, depending on
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TABLE II PURIFICATION OF FOLYLPOLYGLUTAMATE SYNTHASE FROM Lactobacillus casei
Fraction 1. Crude extract 2. DEAE-cellulose 3. 30-60% ammonium sulfate 4. Phosphocellulose P-l 1 5. Phenyl-agarose 6. Sephadex G-100 7. AMP-agarose 8. lO-Aminodecyl-Sepharose
Volume (ml)
Protein (mg)
Specific activity (pmol/hr * mg)
5100 5700
38760 23655
0.00007 0.00013
1.0 1.9
0.00018 0.012 0.23 0.82 2.9 13.8
2.6 170 3300 11700 41000 197000
600 400 65 14 17 8.5
14400 330 9.75 1.36 0.17 0.014
Purification (-fold)
Yield (%I 100 106 87 81 77 38” 17 6.7
0 A higher yield of enzyme (74%) was recovered from the column but only part of this was used in the subsequent steps.
the growth conditions of the bacterial culture, and the purified preparation is homogeneous as judged by sodium dodecyl sulfate-gel electrophoresis. Step I. Crude Extract. All operations are carried out at O-4”. The bacterial suspension is thawed, diluted with 1 volume of 50 miI4 potassium phosphate buffer (PH 7) containing 50 mM KCl, and sonicated for 9 min using a Branson W-350 sonicator in the pulsed mode (30 min at 0.3 s/s). The sonicate is centrifuged and the precipitate resuspended in an equal volume of buffer and resonicated. This process is repeated two more times and the four supernatants obtained are combined to give a crude enzyme extract (Fraction 1). Step 2. DEAE-Cellulose Chromatography. Fraction 1 is applied to a DEAE-cellulose (DE-52, Whatman) column (40 x 7.5 cm) that has been equilibrated with 50 n&I potassium phosphate buffer (pH 7) containing 50 mM KCI. The column is washed with the equilibration buffer (700 ml) and the total flow-through is combined to give Fraction 2. This step is used primarily to remove nucleic acids and polynucleotides, but can result in up to a IO-fold purification when lower amounts of protein are applied to the column. Step 3. Ammonium Sulfate Fractionation. Ammonium sulfate is slowly added to Fraction 2 to give a 30% saturated solution. After stirring for 2 hr, the mixture is centrifuged and the precipitate discarded, Ammonium sulfate is slowly added to the supematant to give a 60% saturated solution and, after stirring for 2 hr, the mixture is centrifuged. The precipitate is resuspended in 50 mM potassium phosphate buffer, pH 7 (500 ml), and dialyzed overnight against the same buffer (40 volumes).
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Step 4. Phosphocellulose Chromatography. The dialyzed extract (Fraction 3) is applied to a phosphocellulose (P- 11, Whatman) column (11.3 x 7.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The column is washed with the equilibration buffer (800 ml) and eluted with a linear gradient (2 liters) of KC1 (O-250 mM) in the same buffer. Fractions containing the bulk of the enzyme activity are pooled (Fraction 4). Most of the applied protein does not bind to the phosphocellulose column. Step 5. Phenyl-Agarose Chromatography. Ammonium sulfate is added to Fraction 4 to give a 70% saturated solution and the mixture is stirred for 90 min and centrifuged. The precipitate is redissolved in 50 mM potassium phosphate buffer (pH 7.5), containing 0.41 it4 ammonium sulfate (10% saturation; pH after addition of ammonium sulfate) (23 ml) and the solution is applied to a phenyl-agarose (BRL) column (9 x 1 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.5), containing 0.41 M ammonium sulfate. The column is washed with buffer/O.41 M ammonium sulfate (30 ml) and buffer/O.33 M ammonium sulfate (30 ml) and eluted with a linear gradient (100 ml) of ammonium sulfate (0.33-o M) in buffer followed by buffer alone. Fractions containing enzyme activity, which elutes in the latter half of the gradient, are pooled. Step 6. Sephadex Chromatography. Fraction 5 enzyme is precipitated with ammonium sulfate (70% saturation) as described above, resuspended in 50 mM potassium phosphate buffer (pH 7), and applied to a Sephadex G-100 column (I 15 x 1.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The column is eluted with the equilibration buffer and fractions containing the majority of the enzyme activity are pooled and dialyzed for 3 hr against two changes of 5 mM potassium phosphate buffer, pH 7 (Fraction 6). Step 7. AMP Affinity Chromatography. Fraction 6 enzyme is applied to an AMP-hexyl-agarose (coupled through N6 of AMP; P-L Biochemicals) column (7 X 1 cm) equilibrated with 5 mM potassium phosphate buffer (pH 7), containing MgC12 (2 m&I). The column is washed with the equilibration buffer (20 ml) and eluted with 50 mM potassium phosphate buffer, pH 7 (15 ml) and 50 mM potassium phosphate buffer (pH 7) containing 250 mM KC1 (15 ml). The fractions with peak activity are pooled and dialyzed for 3 hr against two changes of 50 mM potassium phosphate buffer, pH 7 (Fraction 7). The enzyme is not eluted from this column by MgATP2-, suggesting that binding is due to an ion-exchange or phosphate affinity effect. Under the described conditions, most of the applied protein binds to the column and the enzyme is eluted before the bulk of the protein. Additional studies indicate that enzyme stability and recovery from this column are increased if the enzyme is eluted with 50 mM potassium phosphate buffer (pH 7) containing 10% Me2S0.
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TABLE III PROPERTIESOFBACTERIAL FOLYLPOLYGLUTAMATE SYNTHASW Cotynebacterium
Purification (-fold) pH optimum Monovalent cation Divalent cation Reducing agent M, SDS-gel electrophoresis (Sephadex) Dihydrofolate synthase activity Monoglutamate substrate Polyglutamate substrate Stabilizers
Lactobacillus
7000 9.5 200 mM K+ Mg2+ None
40,000-200,000 9.5 200 mM K+ Mg2+, Mn2+ None
53,000 51,000 Yes
43,000 43,000 No
H$teGlu 5,10-Methylene-H,PteGlu K+, Pi> Me2S0
5, IO-Methylene-H4PteG1u 5,10-Methylene-HrPteGlu K+, P, , Me,SO
a PteGlu, folic acid, pteroylglutamate; H,PteGlu, tetrahydropteroylpoly(-y-glutamate).
Step 8. Aminodecyl-Sepharose Chromatography. Fraction 7 is applied to a IO-aminodecyl-Sepharose column (7 x 1 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7). The bulk of the protein is eluted with the equilibration buffer (30 ml) and the enzyme is specifically eluted with buffer containing 250 mM KCl. Fractions containing enzyme activity are pooled. The purified protein is unstable, presumably due to the low protein concentration. MezSO is added to Fraction 8 enzyme to a final concentration of 20% (v/v) and the extract is divided into l-ml aliquots and stored at -20”. Approximately 50% of the enzyme activity remains after storage for 3 months. Properties
of the Purified
Proteins1*2,7,8
General properties of the purified proteins are listed in Table III. Both proteins are monomeric and have an absolute requirement for a monovalent cation for activity. K+ (200 mM) gives maximal stimulation of activity while Rb+ and NH4+ are active but less effective. The monovalent cation may cause a conformational change in the protein.9 The Mg2+ ’ B. Shane, in “Peptide Antibiotics-Biosynthesis and Functions” (H. Kleinkauf and H. Dohren, eds.), p. 353. de Gruyter, Berlin, 1982. * B. Shane and D. J Cichowicz, Adu. Exp. Med. Biol. 163, 149 (1983). 9 B. Shane, in “Chemistry and Biology of Pteridines” (J. A. Blair, ed.), p. 621. de Gruyter, Berlin, 1983.
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357
SYNTHASE
TABLE IV FOLATESUBSTRATESOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE
SYNTHASE
Relative activity (50 PM substrate) Values of n in Glu-,a
(6s IH&e
HJ’teGlu
(6R)-5,10-MethyleneH,PteGlu
Corynebacterium
(6R)-lo-FormylH,PteGlu
enzyme
20 100
1.5 0 Lactobacillus
46.3 33.3 0.2
11.5 0.4 0
casei enzyme
0.1 1.3 53 6.5
0.1 0.1 0 0
100
0.7
246 79 2.8 3.1 2.8 1.7
7.8 0.4
0.1 0 0 0
0 Subscript to Glu, n, denotes glutamate chain length in H,PteGlu,.
requirement is to generate the MgATP substrate and free ATP is a potent inhibitor of the reaction. The high pH optimum reflects the pK of the amino group of the glutamate substrate. The free amine form is the substrate for the reaction, and the reaction proceeds efficiently at pH 8 provided high levels of glutamate are provided. One mole of ATP is hydrolyzed to ADP and Pi for every mole of glutamate added to the folate or pteroate substrates. The folate substrate specificities of the enzymes are shown in Table IV. The Corynebacterium protein will use dihydropteroate and tetrahydrofolate as substrates. However, 5,10-methylene derivatives are the preferred polyglutamate substrates. The purified protein will metabolize labeled 5, lo-methylenetetrahydrofolate to the tetraglutamate derivative, which is the predominant polyglutamate found in viuo. The Lactobacillus enzyme will not utilize dihydropteroate as a substrate and preferentially uses 5,10-methylenetetrahydrofolate mono- and polyglutamate derivatives as its substrates. The purified L. casei enzyme will metabolize labeled 5, lo-methylenetetrahydrofolate primarily to the tetra- and pentaglutamate derivatives and small amounts of longer glutamate chain length derivatives are formed. The major in uiuo folates in Lactobacillus are octa- and nonaglutamate derivatives.
358 KINETIC
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TABLE V CONSTANTSOFFOLYLPOLYGLUTAMATESYNTHASE-DIHYDROFOLATE~YNTHASE
Wd Compound
(pmol/hr . mg) Cotynebacterium folylpolyglutamate synthetase
MgATP (6&Y)-H$teGlu L-Glutamate Phosphate p, y-Methylene-ATP ATP-y-S
18.0 2.1 160 -
10.0 12.7
MgATP HJ%eGlu L-Glutamate 8, y-Methylene-ATP ATP-y-S
Corynebacterium dihydrofolate synthetaseb 2.9 co.4 1380 1.0 0.29
4 750 0.58 3.0
Lactobacillus casei folylpolyglutamate synthetase MgATP 5600 800 2.3 15 (6R)-5,10-Methylene-H,PteGlu2 L-Glutamate 423 Phosphate 12,500 & y-Methylene-ATP 597 ATP-y-S 105
45.1 45.4 41.2
-
-
64.9 67.5 62.3 -
-
0 Not estimated or not applicable b Approximate values; detailed kinetic analyses not carried out.
The kinetic mechanism for both folylpolyglutamate synthases is ordered ter ter with the nucleotide substrate binding first, followed by the folate and glutamate substrates, and the order of product release is ADP, folate product, and Pi. z*OThis mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. A catalytic mechanism involving the formation of a folyl-y-glutamylphosphate intermediate followed by a nucleophilic attack by the free amine of glutamate on this mixed anhydride is suggested. Kinetic constants for the enzymes are shown in Table V. Dihydropteroate is a noncompetitive inhibitor of the Corynebacteriumfolylpolyghttamate synthaseactivity while tetrahydrofolate does not significantly inhibit the dihydrofolate synthase activity, suggestingthat the protein is bifunctional. The affinities of nucleotide analog inhibitors differ for the two I0 B. Shane, J. Biol. Chem. 255, 5663 (1980).
[551
FOLYLPOLYGLUTAMATE
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359
Corynebacterium synthase activities. As the nucleotide binds to free enzyme, this also suggests that the protein is bifunctional. Kinetic constants for the Lactobacillus folylpolyglutamate synthase are similar except for a greatly decreased afiinity for the nucleotide substrate, which is also reflected by a decreased affinity for nucleotide inhibitors. It should be noted that under the conditions of the standard assay, glutamate (250 PM) is not saturating for any of the activities and ATP (5 mM) is not saturating for the Lactobacillus enzyme. ATP is the preferred nucleotide substrate of both proteins although other purine triphosphates and UTP will substitute to some extent. ZTP is also a substrate for the Lactobacillus enzyme. The glutamate binding site is specific for L-glutamate although L-homocysteate and 4-fluoroglutamate are alternative substrates. Other Purified Folylpolyglutamate
Synthases
The E. coli folylpolyglutamate synthase-dihydrofolate synthase gene has recently been cloned and amplified and the protein has been purified to homogeneity.3 Its properties are similar to the Corynebacterium protein. It appears to be bifunctional and utilizes 5, lo-methylene derivatives as its polyglutamate substrate. The preferred monoglutamate substrate is lo-formyltetrahydrofolate.**J* The hog liver folylpolyglutamate synthase has been purified to homogeneity.13It has a slightly higher Kcatvalue than the bacterial enzymes. In contrast to the bacterial enzymes, the mammalian enzyme has an absolute requirement for a reducing agent, it can utilize oxidized folates as substrates, and the preferred polyglutamate substrates are tetrahydrofolate derivatives. Acknowledgment Supported in part by PHS Grant CA 22717 and Research Career Development Award CA 00697 (B.S.) from the National Cancer Institute, Department of Health and Human Services.
I’ M. Masurekar and G. M. Brown, Biochemistry 14, 2424 (1975). I* R. Ferone, S. C. Singer, M. H. Hanlon, and S. Roland, in “Chemistry and Biology of Pteridines” (J. A. Blair, ed.), p. 585. de Cruyter, Berlin, 1983. I3 D. J. Cichowicz and B. Shane, in preparation.