2256
Biochemistry 1986, 25, 2256-2264
Tewey, K. M., Chen, G. L., Nelson, E. M., & Liu, L. F. (1984a) J . Biol. Chem. 259, 9182-9187. Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D., & Liu, L. F. (1984b) Science (Washington, D.C.) 226, 466-468. Tricoli, J. V., Sahai, B. M., McCormick, P. J., Jarlinski, S. J., Bertram, J. S., & Kowalski, D. (1985) Exp. Cell Res. 158, 1-14.
Tse-Dinh, Y.-C., Wong, T. W., & Goldberg, A. R. (1984) Nature (London) 312, 785-786. Wang, J. C. (1985) Annu. Rev. Biochem. 54, 665-697. Wozniak, A. J., & Ross, W. E. (1983) Cancer Res. 43, 120-124. Wozniak, A. J., Glisson, B. S., Hande, K. R., & Ross, W. E. (1984) Cancer Res. 44, 626-632.
Substrate Specificity of Formylglycinamidine Synthetase? F. J. Schendel and J. Stubbe* Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Received September 25, 1985; Revised Manuscript Received December 2, 1985
Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and A T P to FGAM, ADP, glutamate, and PI, has been purified to homogeneity (sp act. 0.20 pmol min-' mg-I) from chicken liver by an alternative procedure to that of Buchanan et al. [Buchanan, J. M., Ohnoki, S., & Hong, B. S. (1978) Methods Enzymol. 51, 193-2011 (sp act. 0.12 pmol min-' mg-I). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm ( R = C H 2 N H C H O ) has been replaced by R = CH,, C H 2 0 H , CH,Cl, CH2NH3,CH2NHCOCH3,CH2NHCOCH2Cl,CH2NHC02CH2Ph,and L-CHCH,NHCHO. These compounds have been characterized by 'H and 13CN M R spectroscopy. With compounds R = CH,, C H 2 0 H , and C H 2 N H C O C H 3 and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of PI at 4.5, 48, and 20%, respectively, the rate of production of PI from formylglycinamide ribonucleotide. Only R = CH2NHCOCH, causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = C H 2 N H C H O ) and the FGAR analogue (R = CH,NHCHOCH3) in the presence of A T P and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. [ 'sO]-/3-FGAR was prepared biosynthetically, and FGAM synthetase was shown by ,'P N M R spectroscopy to catalyze the transfer of amide to inorganic phosphate. ABSTRACT:
Elegant studies by Buchanan and co-workers have elucidated the role of glutamine in this enzyme-catalyzed amidine production. Their studies with [ ''C]glutamine indicated that in the absence of other substrates the enzyme forms a 1:l complex with glutamine ( t , j z= 125 min). Futher studies indicate that NH LNHCHO a covalent thio ester of glutamine is formed enzymatically with liberation of "NH3". @ w N H L N H C (H 1) o g l p >This "NH3" is the putative nucleophile in the FGAR FGAM conversion and remains associated HO OH with a specific locus in the active site. Hydrolysis of the ATP A D P + PI FGAR FGAM enzyme glutamyl moiety in the absence of other substrates is 0.5% the rate of turnover for the overall reaction (Mizobuchi enzyme has been previously purified 300-fold to homogeneity & Buchanan, 1968b). from chicken liver and found to consist of a single polypeptide Intriguingly, a stable complex is also formed between ATP, chain of M , 133000 (Mizobuchi & Buchanan, 1968a; BuFGAR, and enzyme with a stoichiometry of 0.7 mol of subchanan et al., 1978). Many detailed mechanistic studies have strate/mol of enzyme in the absence of glutamine ( t l j 2= 62 been undertaken by Buchanan and co-workers and have been min at 0 "C). Furthermore, there is evidence that the terminal recently reviewed (Buchanan, 1982). FGAM synthetase has phosphoanhydride bond of ATP is cleaved to ADP and a also been purified 56-fold from Erlich ascites tumor cells by phosphate moiety (perhaps E-P03) during this complex Chu and Henderson (1972). Mechanistic studies on this formation (Mizobuchi et al., 1968). partially purified protein indicate considerable differences between it and the liver enzyme. Formylglycinamidine ribonucleotide (FGAM)' synthetase catalyzes the fourth step in the purine biosynthetic pathway conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, P,, and ADP (eq 1). This
95% by I3C NMR), 10 pmol of [13C,160]glycine(Hyman, 1957), 1.0 pCi of [l-14C]glycine(sp act. 49.3 mCi/mmol), 100 pmol of ATP, 100 pmol of PEP, 400 pmol of MgCl,, 30 pmol of folinic acid, 2 pmol of azaserine, and 5 units of pyruvate kinase in 10 mL of 50 mM Tris-HC1 (pH 8.0) was added 10 mL of
VOL. 25, NO. 8, 1986
2259
the purine biosynthetic enzymes, 400 mg of protein. The reaction mixture was incubated at 37 OC for 90 min and then quenched with 1 mL of 60% TCA. The precipitated protein was removed by centrifugation and the pellet washed 3 times with 5 mL of 5% TCA. The combined supernatants were diluted to 300 mL with water, adjusted to pH 8.0 with 3 N KOH, and then applied to a DEAE-Sephadex A-25 column (2.5 X 18 cm). The column was washed with 2 volumes of water, and the FGAR was eluted with an 800-mL linear gradient from 0 to 400 mM triethylammonium bicarbonate. The fractions containing FGAR eluted at 200 mM triethylammonium bicarbonate and were located by scintillation counting, pooled, and evaporated to dryness in vacuo. The FGAR was redissolved in 10 mL of water, the pH adjusted to 8.5 with 3 N KOH, and 200 pmol of barium bromide added. The precipitated barium phosphate that formed was removed by centrifugation and the pH of the supernatant readjusted to 8.5. Five volumes of cold absolute ethanol was added and the solution placed at -20 OC for 4 h. The precipitate that formed was collected, dried over P,05 in vacuo, and dissolved in water, and the barium salt was exchanged for sodium by passage through Dowex 50W-X8 (Na+ form): yield 10 pmol; proton NMR (D20, pD 6.0) 6 3.69 (d, 2 H, Ji3C-H = 4.8 Hz, CH, of formylglycine), 5.14, (dd, 1 H, Ji3CNCH = 2.0 Hz, JIt-,, = 5.0 Hz, 1'), 7.86, (s, 1 H), formyl proton). "0 Transfer Experiment. To a 10-mm N M R tube containing 60 pmol of ATP, 60 pmol of L-glutamine, 240 pmol of KCl, 120 pmol of MgC12, and 100 pmol of Tris-HC1, pH 8.0, in a final volume of 2 mL (20% D,O) was added 0.5 unit of FGAM synthetase (0.20 unit/mg). The 31PNMR spectrum was taken to 80 MHz, and then 6.5 pmol of [180]FGAR was added and the tube incubated at 37 OC for 30 min. When the reaction was complete, as determined by 31PNMR, the reaction mixture was diluted to 25 mL with water and passed through an Amicon ultrafiltration apparatus with a PM-30 membrane. The filtrate was collected and applied to a DEAE-Sephadex A-25 column (0.5 X 4 cm). The column was washed with 2 volumes of water, and the inorganic phosphate was eluted with 200 mM triethylammonium bicarbonate (pH 7.8). The fractions containing the inorganic phosphate were pooled, concentrated to dryness in vacuo, and subjected to 31P N M R (80-MHz) analysis. The ratio of '60:180-labeledPi produced was determined by weighing the appropriate 31P peaks and found to be 51:49, respectively. A sweep width (f1000 Hz), quadrature phase detection, a 90' pulse angle, and a 16K data block were used. The acquisition time was 3.4 s, and the pulse delay was 2 s. To enhance resolution, exponential multiplication with a line-broadening factor of 0.1 Hz was applied to the FID before Fourier transformation. Nature of ATPase by I . The reaction was carried out in a 5-mm NMR tube with a final volume of 1.O mL (30% DzO) containing 10 mM Tris-HC1 (pH 8.0), 80 mM KCl, 20 mM MgCl,, 60 mM ATP, 40 mM alp-I, 8 units of FGAM synthetase, and 500 pL of HZ1'0 (I8Ocontent 45.1%). The reaction was incubated at 37 OC and followed by 31PNMR. After the reaction had produced between 5 and 10 pmol of inorganic phosphate, the enzyme was removed by ultrafiltration through a PM-30 membrane with an Amicon ultrafiltration apparatus. The inorganic phosphate was isolated by DEAE-Sephadex chromatography and subjected to 31PNMR analysis as described above. ATP, FGAM Synthetase, FGAR, or VI Complex. The incubations were carried out in a total volume of 400 pL containing 25 mM potassium phosphate (pH 6.5), 20 mM MgCl,, 80 mM KCl, 20 mM [y32P]ATP(2.8 X lo6 cpm/
2260
BIOCHEMISTRY
SCHENDEL A N D STUBBE
Table I: Isolation of FGAM Synthetase
met hod crudea
(NH4)2so4
acid
(NH4)2SOo DEAE-Sephadex
total protein (mg) 961 1 3 200 1320 840 132
protein (mg/mL)
total act. (units)
sp act. (uni ts/mg of protein)
33 16 6.5 4.0 0.4
1.8 3.2 6.6 8.4 6.6
0.002 0.00 1 0.005 0.0 1 0.05
3.7 9.7
0.20 0.05
A-SO
hydroxyla pat i t e DEAE-Sephadex
18.5 180
0.29
0.45
A-50* CM Sephadex C-50 47 0.5 4.8 0.10 167 0.54 7.5 0.05 A-5W Sephacryl S-200 24 0.95 4.0 0.16 " A total of 75 g of chicken liver. different preparation starting with 100 g of chicken liver. C Athird preparation starting with 100 g of
chicken liver.
pmol), 0.05 unit of FGAM synthetase, and 9 mM of either a/&FGAR or FGAR analogue. The reactions were incubated at 37 OC for 5 min and then applied to a Sephadex G-50 column (0.75 X 21 cm) equilibrated in 100 mM potassium phosphate, pH 6.5. Fractions of 1 mL were collected, the absorbance at 280 nm was recorded, and 500 p L of each fraction was analyzed for 32Pin a liquid scintillation counter. The molar ratio of [ T - ~ ~ P ] A Tbound P to the enzyme was calculated with 196000 M-' cm-I as the extinction coefficient for the enzyme at 280 nm (Mizobuchi et al., 1968). RESULTSA N D DISCUSSION FGAM Synthetase Pwifcation. The most recent purification of FGAM synthetase (Ohnoki et al., 1977; Buchanan et a!., 1968), a modification of the original procedure of Mizobuchi and Buchanan (1968a), reports isolation of 57 mg of protein of specific activity 0.12 pmol mi& mg-' in 24% overall yield, starting with 475 g of chicken liver.3 We encountered a number of difficulties in attempting to reproduce this isolation and therefore developed an alternative procedure as indicated in Table I. This procedure gives 18.5 mg of protein of specific activity 0.20 pmol min-' mg-l in 43% overall yield starting with 74 g of chicken liver. The enzyme appears to be =90% pure on the basis of SDS gel electrophoresis (Figure 1) and has a M, 133000, as reported earlier. Antibodies prepared to homogeneous FGAM synthetase and used in Western blot studies indicate that the 5-10% contaminating proteins are cross-reactive with the antibody and, hence, most probably are proteolytic fragments of FGAM synthetase. It should be noted that Buchanan's specific activities, as well as our own, are based on the conversion of the FGAM produced during this reaction to aminoimidazole ribonucleotide (AIR) with AIR synthetase. We have recently purified this enzyme to homogeneity from E. coli and chicken liver (Schrimsher et al., 1986).2 Hence, our coupling enzyme does not possess the problems that might be encountered with the ethanol fraction of pigeon liver possessing AIR synthetase used by earlier workers (Flaks & Lukens, 1963). The isolation procedure described in Table I for purification of FGAM synthetase represents the most reproducible and simplest methodology developed to date. While we purified Buchanan's units have been defined as "that amount which causes a change of 0.1 of an absorbance unit at 500 nm per 20 min incubation in the AIR synthetase assay." One such unit is equivalent to 0.00029 standard unit. Therefore, pure protein with specific activity of 380 units is equivalent to 0.12 pmollmin.
1 2 3 FIGURE1 : A 7.5% SDS gel of FGAM synthetase isolated as described under Materials and Methods: (lane 1 ) 60 pg of protein from the
hydroxylapatite column; (lane 2) 35 pg of protein from the Sephacryl S-200 column; (lane 3) 52 pg of protein from the CM-Sephadex 50 column. These correspond to the three isolation procedures shown in Table 1.
this protein to apparent homogeneity by a variety of other procedures, the reproducibility depended on the time of year we obtained the chicken livers, as well as the rapidity of processing the livers. This alternative procedure was developed as several problems were encountered with the earlier procedures described by Buchanan and co-workers. (1) Their preparation of the acetone powder requires use of 19 L of acetone at -20 OC for 4.5 kg of chicken. The problems with the ventilation of these acetone vapors in a cold room were insurmountable. This step was therefore eliminated. (2) In our hands, FGAM synthetase activity either did not bind to the DE-52 column or, when it did bind, was eluted as multiple peaks. In fact, French et al. (1963) studying the Salmonella typhimurium FGAM synthetase reported similar problems with multiple peaks of activity. Therefore, a DEAE-Sephadex A-50 replaced the DE-52 anion-exchange columns. The method we now report is rapid and has been quite reproducible. Furthermore, the last hydroxylapatite column can be replaced by either a sizing column such as Sephacryl G-200 or a cation-exchange column such as CM-Sephadex C-50 to produce protein of specific activity ranging from 0.10 to 0.20 pmol min-' mg-I (Figure 1, lanes 1-3). Stability of FGAM Synthetase. This enzyme is reasonably selective about its environment. Mizobuchi and Buchanan in 1968 reported that the enzyme in Tris buffer at pH 7.2 lost 40% of its activity in 100 min at 0 "C; we have made similar observations with HEPES buffer (pH 7.2). The higher the pH, the more rapid the loss in activity. The enzyme is therefore routinely stored in 20 mM phosphate (pH 6.5). Furthermore, as reported by Ohnoki et al. (1977), 1 mM glutamine and 10% glycerol also greatly stabilize FGAM synthetase activity. The enzyme stored under these conditions retains full activity after 2 months at -30 OC and is routinely stored in small aliquots due to loss of activity during freezing and thawing. Synthesis and NMR Characterization of FGAR. Several chemical syntheses of FGAR have been previously reported (Carrington, 1965; Chu & Henderson, 1970). We have used a modification of the procedure of Chu and Henderson to prepare FGAR and a variety of FGAR analogues. This procedure (Scheme I) produces a 1:l mixture of a- and 8anomers. Furthermore, the assignment by Chu and Henderson of the downfield doublet at 5.70 ppm to the @-anomericproton of FGAR and the upfield doublet at 5.40 ppm to the a-
2261
VOL. 2 5 , NO. 8 , 1986
F G A M S Y N T H E T A S E S U B S T R A T E SPECIFICITY Scheme I
Table 11: Anomeric Chemical Shifts of FGAR” ~~
compd
FGAR~
H-I’ (ppm)
J , L ~ (Hz) ,
formyl (ppm)
5.08, 5.37 5.09 5.39
5.3, 4.6 5.3 4.5
7.89
I
7.85 FGAR‘ 7.86 FGAR~ “Chemical shifts are referenced to HOD at 4.40 ppm. bFGAR prepared by the synthetic method described under Materials and Methods. FGAR prepared by the biosynthetic method described under Materials and Methods. This material is enzymatically active. d A 1:l anomeric mixture of FGAR was incubated with FGAM synthetase. At the end of the reaction, FGAM was separated from remaining FGAR, which was not a substrate for the synthase.
anomeric proton of FGAR is incorrect (Table 11). We have assigned the upfield anomeric proton to 0-FGAR on the basis of its identity with the @-FGAR we have prepared biosynthetically by modifications of the procedure of Lukens and Flaks (1963). Furthermore, incubation of a@-FGARwith FGAM synthetase results in recovery of 0.5 equiv of FGAM and 0.5 equiv of unreacted a-FGAR (downfield anomeric proton) (Table 11). Preparation of FGAR Analogues. Early studies by Mizobuchi et al. (1968) reported that FGAR, ATP, and FGAM synthetase formed a complex, isolable by Sephadex G-25 chromatography, with a half-life of 62 min at 0 “C. We hoped to take advantage of this unique complex formation by making an FGAR affinity column that in the presence of ATP would allow rapid isolation of the protein. Furthermore, Li and Buchanan (197 1) reported that N-(bromoacety1)glycinamide was a potent inactivator of FGAM synthetase. Therefore, to investigate the nature of the FGAR binding site, to design a potential affinity ligand, and to prepare active site directed affinity labels, we undertook the synthesis of a variety of FGAR analogues (Table 111). The general synthetic approach is analogous to that of Chu and Henderson and is indicated in Scheme I. Interestingly, the synthetic procedure outlined in Scheme I produces an a@ (1:l) anomeric mixture of nucleosides. These are easily separable by low-pressure silica gel chromatography at the benzoylated sugar stage. Removal of the blocking groups allows isolation of pure a-and P-nucleoside. A wide variety of phosphorylating agents have been investigated for conversion of the nucleoside to nucleotide. The only method that worked successfully, however, was the one described by Chu and Henderson, a modification of the procedure of Yoshikawa et al. (1967), using a 10-fold excess of P0Cl3. Under these conditions the anomerically pure nucleosides are Table 111: Anomeric Chemical Shifts of FGAR Analogues” compd R anomer
BZO
BzO
0
BzO
OBz
BzO
0
OBz
HO
PO(OEt ),
- og0Y7NHAR HO
OH
converted to a 1:l anomeric mixture of nucleotides. The proton N M R data of these analogues are summarized in Table 111. The downfield anomeric proton of all the FGAR analogues possesses a smaller 1’-2’ coupling constant and has been assigned to the a-anomeric configuration. This assignment is based on the comparison of the anomeric region of @-GAR and P-FGAR prepared biosynthetically with a 1:l mixture of ab-GAR and a@-FGARprepared synthetically. In both cases, the downfield, biochemically inactive, a-anomer possesses a smaller 1’-2’ coupling constant than that observed for the biochemically active upfield @-anomer. The 13CN M R data of these analogues are summarized in Table IV. The assignment of I3C resonances is based on the established order of C-l’, C-4’, C-3’, C-2’, and (2-5’ in the direction of increasing field with the assignments of Chettur and Benkovic (1977) for &GAR (IV) and uridine 5’monophosphate as reference material (Dorman & Roberts, 1970). Interestingly, in the case of the a isomer the normal C-1’ and (2-4’ relationship is reversed with C-4’ being located downfield from the C-1’. Assignments of C-4’ are easily made due to coupling with the phosphate moiety. Compounds I-IX were incubated with FGAM synthetase and assayed with either the [y-32P]ATPand [32P]P,determination or the glutamate dehydrogenase and 3-acetylpyridine adenine dinucleotide reduction assay described under Materials and Methods. The results of the [32P]PIassay are summarized in Table V. Three of the compounds (R = CH20H, CH,,
J,,_zf(Hz)
methyl (ppm)
P
5.01
5.2 4.8 5.0 4.6 4.8 4.4 5.3 4.4 5.3 4.6 5.3 4.5 5.2 4.4 4.4 4.0
1.82 1.89
a
5.12 5.38 5.08 5.32
11
CH2OH
P
111
CH2C1
a
5.28 5.09 5.38 5.08 5.37 5.05 5.32 5.08 5.37 5.15 5.40 5.08 5.32
P
V
CH2NHCHO
P
VI
CH2NHCOCH3
P
VI1
CH2NHCOCH2CI
P
VI11
CH2NHCOOCHzPh
P
IX
L-CHCH~NHCHO
P
a a a a a 01
OH
0
POC Is
€3-1’ ( P P d
CH2NH3*
c
DCC
0 B ~ O ~ N H II- C CH, R ON^
P
IV
I1
R-C-0-
Not Isolated
CH3
a
I
OBz
0
B z o P N t i z
I
“Chemical shifts are referenced to HOD at 4.40 ppm.
I
I
OB2
5.0 4.5
other (ppm)
7.89 1.62 1.65
1.oo
7.68
2262
B IO C H E M IS T R Y
SCHENDEL AND STUBBE
Table IV: ')C Chemical Shifts of FGAR Analogues C-1' C-2' compd" R (PPm) (PPm) I CHI 82.55 70.20' 79.36 69.28 I1 CHZOH 82.53 70.20' 78.72 69.32 111 CH,CI 83.28 69.64' 79.66 69.24 lVb CH2NHI 83.30 70.75' 69.85 79.76 V CH,NHCHO 83.54 70.73' 79.92 69.86 VI CH,NHCOCH, 82.80 69.99' 79.31 69.21 VI1 CH,NHCOCH,CI 83.39 73.53' 79.89 70.33 VI11 CH,NHCO,CH,Ph 83.12 70.32' 79.40 69.45
C-3' (PPm) 72.97 70.25' 72.96 69.98' 69.98 72.88 73.64 70.75' 73.66 70.81' 70.09 72.91' 70.56 69.93' 70.49 73.28'
C-4' (PPd 82.39 80.44 82.34 81.46 81.46 80.27 83.15 81.36 83.15 81.53 82.07 80.71 82.74 81.31 82.62 81.33
Jc.&-p
(Hz) 8.3 8.3 9.6 8.2 8.4 8.2 8.6 8.6 8.3 8.2 7.9 7.9 9.0 6.0 8.7 8.3
C-5' (PPm) 63.00d
Jc.5,-p (Hz) 3.85
63.09
3.0
64.45
4.6
63.53
3.3
63.62
2.9
63.41
3.7
amide (PPm) 174.57 174.13 175.36 174.86 169.75 169.33 173.47 174.73 171.66 171.34 171.26 171.65
64.02 63.01
5.1
173.21 173.63
methylene (PPm)
other (PPm) 21.43 21.39
60.28 41.69 41.53 42.62 42.42 41.10 40.96 41.91 41.72 41.67 42.53 43.32 43.05 66.72
164.71 20.96 174.09 20.91 128.88 156.71
I3C NMR spectra were taken at 50 MHz with broad-band proton decoupling. Chemical shifts are referenced to an external standard dioxane at 66.50 ppm. All samples were run in D20at pD 6.0. For each compound, the top row represents the chemical shifts of the 0 compound while the second row represents the chemical shifts of the a compound. *GAR has previously been prepared by Chettur and Benkovic (1977). 'The chemical shift of the C-2' of the @-isomer is not distinguishable from the chemical shift of the (2-3' of the a-isomer. In some cases an anomeric distribution of a:P that varied significantly from 1:l allowed the assignment to be made. dOnly one peak was observed for the C-5' carbon of both a- and @-anomers. Table V: ATPase Activity of FGAR Analogues with FGAM Synthetase
4a-v
I1 VI I I11 IV VI1 VI11
IX
CH~NHCHO~ CH,OHC CH,NHCOCH3' CHI' CH2C1 CH2NH3 CH2NHCOCH2Cl CH2NHCOZCH2Ph 1.-CHCH,NHCHO
0.18 2.4 6.6 2.3
124 4.6 0.65 0.44
99 48 20 4.5
Prepared biosynthetically. *Prepared by the chemical procedure as described under Materials and Methods.
