- Email: [email protected]
Photooxidation of formyltetrahydrofolate synthetase in the presence of methylene blue
ARCHIVES
OF
BIOCHEMISTRY
AND
Photooxidation
BIOPHYSICS
E. MACKENZIE; Department
421-427
(1972)
of Formyltetrahydrofolate Presence
ROBERT
160,
LINDA
of Biochemistry,
Synthetase
of Methylene D’ARI
University
in the
Blue’
STRAUS,
AND
JESSE C. RABINOWITZ
of CaKforrGa, Berkeley,
California
94720
Received February 2, 1972; accepted March 3, 1972 Photooxidation of formyltetrahydrofolate synthetase from Clostridium acidi-urici in the presence of methylene blue results in a pseudo-first-order loss of enzymic activity and destruction of histidine residues. In addition, a loss of cysteine and methionine residues occurs at levels of inactivation greater than 50%. Both the pH rate profle for photoinactivation which shows an inflection point near pH 6.3, as well as the correlation of loss of activity with the loss of two histidines per subunit, indicate that the primary event responsible for inactivation is the destruction of histidine. Measurements of the ability of the photooxidized enzyme to bind substrates using the method of equilibrium partitioning indicate that the loss in activity is not due to an inability to bind ATP or tetrahydropteroyl triglutamate, and thus a possible catalytic role for histidine is suggested.
The enzyme thetase (EC
cylindrosporum
formyltetrahydrofolate
syn-
6.3.4.3) from Clostridium and Clostridium acidi-urici
has been extensively investigated, but its mechanism of action is not understood. Early studies of partial exchange reactions (1) indicated that the enzyme operates by a concerted mechanism and subsequent kinetic
analyses (2, 3) and measurements of equilibrium exchange rates (4) have been consistent with that proposal, although mechanisms involving enzyme bound covalent intermediates cannot be ruled out (5). Recent
studies have shown that the enzyme
is composed of four apparently identical subunits by physical-chemical criteria (6) and that four nucleotides and four moles of tetrahydropteroyl triglutamate bind per mole of enzyme (5, 7). The tetrameric enzyme can be dissociated in the absence of certain monovalent cations to inactive
monomers (8, 9) that bind nucleotides but not tetrahydropteroyl triglutamate (10). However, the types of amino acid residues involved in substrate binding, catalysis, and the cation-dependent subunit interaction that results in formation of the tetrahydropteroyl triglutamate binding site are unknown. This investigation was undertaken to determine the nature of amino acid residues required for activity of the enzyme as well as to attempt to elucidate their role. Photooxidation of formyltetrahydrofolate synthetase from 6. acidi-urici with methylene blue results in a rapid loss of enzymic activity that correlates with the destruction of two histidine residues per subunit. MATERIALS
a The 421
@ 1972 by Academic
Press,
Inc.
METHODS
Materials. IO-Formyltetrahydrofolate synthetase was purified from C. acidi-urici by a modification of a procedure previously described (11). The modifications include chromatography on BioGel and DEAE-cellulose columns and the procedure yields a crystalline enzyme with a specific activity of 360-380 units per mg. The enzymic activity was assayed as previously described (11) .3 The sodium salt of ATP was a prod-
1 This work was supported in part by Grant AM-2109 from the National Institutes of Health, United States Public Health Service. 2 Present address: Department of Biochemistry, McGill University, Montreal 110, Quebec, Canada.
Copyright
AND
concentration
of MgClz
used is 0.1
M.
422
MACKENZIE.
STRAUS.
uct of Sigma. dl-Tetrahydrofolic acid was prepared by catalytic reducion of folic acid in aqueous solution (12) followed by purification on DEAE-cellulose (13). [aH]adenosine (G)d’-triphosphate (7.93 Ci/mmole) in 50y0 ethanol was obtained from New England Nuclear and was lyophilieed before use. Pteroyltriglutamate was a generous gift of Dr. L. Ellenbogen of Lederle Laboratories and was used in the preparation of Z-[6,7-aH2]tetrahydrofolate as described by Curthoys, Scott and Rabinowits (14) using [3H]sodium borohydride (590 mCi/mmole) from Amersham Searle. Methylene blue was a product of the National Aniline Division, Allied Chemical Corporation and ultra pure guanidine hydrochloride was purchased from Mann. Omnifluor was obtained from New England Nuclear Corp., and BiosolvBBS-3 from Beckman. All other chemicals were reagent grade. Photoinactivation. Crystals of enzyme were collected from ammonium sulfate suspensions by centrifugation, dissolved in the appropriate amount of 50 mM Tris chloride buffer (pH 7.8) containing 50 rnM KCl, and dialyzed overnight against three 500 ml changes of the same buffer. Protein concentration was determined using E%o = 8.88 X lo4 cm2/mmole (5). Photooxidation was carried out with an apparatus described by Ray (15) using flasks with optical glass bottoms for large volumes (l-2 ml) of enzyme for subsequent use in binding studies. Smaller volumes of 300 ~1 were photooxidized in 12 X 100 mm Pyrex test tubes. The temperature was maintained at 20” and the tubes were shaken by hand at one minute intervals. In kinetic experiments, 10 ~1 aliquots were removed at various times and diluted into 10 ml of 50 InM Tris chloride buffer (pH 7.8) containing 50 IIIM KCl. Aliquots (1Orrli of this diluted enzyme were assayed for activity (11). Solutions of methylene blue were made on a weight basis, and due to the instability of this dye, the concentrations quoted represent maximal values. Amino acid analysis. Samples of photoinactivated enzyme were dialyzed overnight against 1 liter of 10 mM potassium phosphate (pH 7.8) in the dark. Acid hydrolysis of the samples was carried out in evacuated tubes with 6 N HCI for 24 hr at 110”. Tryptophan was determined spectrophotometrically (16) and methionine after alkaline hydrolysis (17) in 12 X 75 mm Nalgene tubes sealed with serum caps. The tubes were evacuated and flushed with nitrogen several times before hydrolysis at 110” for 16 hr. Analyses This value was inadvertently original description.
omitted
from
the
AND
RABINOWITZ
were carried out with t,he Beckman Model 117 Automatic Amino Acid Analyzer by the singlecolumn method. Amino acid compositions were calculated relative to glutamic acid in the case of acid hydrolysis or leucine with alkaline hydrolysis. Cysteine was measured using 5,5’-dithiobis-(2nitrobenzoic acid) (18) in 6 M guanidine hydrochloride containing 1 mM EDTA and 0.008% DTNB. Absorbance at 412 nm was measured immediately after mixing. Since methylene blue did not interfere with the determination of sulfhydryl groups, the dialysis step was eliminated in this case. However, the preparation of samples was carried out in a darkened room to minimize further photooxidation. Binding of ligands. The binding of ligands as measured by two-phase partitioning w&s carried out as described by Curthoys and Rabinowitz (5, 7). Methylene blue was found not to interfere and was not removed from samples. Enzyme was used directly following photooxidation for tetrahydropteroyltriglutamate binding experiments, but was concentrated approximately IO-fold by vacuum dialysis prior to use by measuring ATP binding. Although tetrahydrofolic acid is regularly used in assaying the enzymic activity, the natural substrate, I-tetrahydropteroyltriglutamate, has greater affinity for the enzyme (7) and consequently was preferred for binding experiments. Radioactivity was measured by use of a toluene, Omnifluor, and Biosolv-BBS-3 scintillation solutions (5, 7). RESULTS
The pseudo-first-order rates of photoinactivation of formyltetrahydrofolate synthetase at three concentrations of methylene blue are shown in the semilogarithmic plot in Fig. 1. Over the range of about 0.84 moles of methylene blue per mole of enzyme, the rate of inactivation was linearly dependent upon the dye concentration. A ratio of 2 moles of methylene blue per mole of enzyme resulted in a convenient rate and was generally used in the remainder of the experiments reported here. ATP did not affect the rate of inactivation and tetrahydrofolic acid could not be tested due to its own susceptibility of oxidation. The pH rate profile for the photoinactivation process is shown in Fig. 2. Although the enzyme is less stable at pH values below 6 (19)) no loss in enzymic activity was observed during the time necessary to obtain initial rates of photoinactivation. No photo-
FORMYLTETRAHYDROFOLATE
Minutes
FIG. 1. Pseudo-first-order rate of photoinactivation of formyltetrahydrofolate synthetase. Protein was 3 mg/ml (1.25 X 1W6 M) in 50 mM Tris chloride buffer (pH 7.8) containing 50 mM KCl. Methylene blue was 1 X 1W6 M (0). 2.5 X W5 M (0) and 5 X 1W M (A).
about 20% of its original activity, followed by amino acid analysis, showed that of the photooxidizable residues (20) only histidine, methionine and a small amount of cysteine are destroyed as shown in Table I. Complete amino acid analysis showed no differences in the amounts of other amino acids. Since analysis of acid hydrolysates showed that tyrosine was not destroyed, a value of 9 residues per subunit was used in calculating the tryptophan content (16). Figure 3 and Table I show that no significant loss of sulfhydryl groups or methionine occurs until about 50% inactivation, which clearly eliminates these residues as primary targets of the photooxidation. However, a linear correlation was found between loss of activity and histidine destruction as shown TABLE
* . rate or pnotoinactivation. Final concentrations were 1.25 X lo+ M enayme, 2.5 X lO+ M methylene blue in 0.25 M potassium succinate buffers. The pH was adjusted immediately prior to inactivation. Initial rates were obtained from semilogarithmic plots of activity versus time.
inactivation was observed below pH 5.5 and a maximal rate was obtained above pH 7. The shape of this curve with an inflection point at pH 6.3 implicates the destruction of histidine as the event responsible for loss of enzymic activity. Photoinactivation of the synthetase to
I
COMPOSITION OF FORMYLTETRAHYDROFOLATESYNTHETASE AFTER PHOTOXIDATION WITH METHYLENE BLUE
AMINO
ACID
Amino acid
FIG. 2. Effect of pH on the initial
423
SYNTHETASE
Residues per monomeric unit’ Control Photooxidized*
photosensitive Histidine Tyrosine Cysteine Tryptophan” Methionine
11.3 f 9.2 f 6.1 2.3 11.2 f
0.1 0.1
photostable Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Isoleucine Leucine Phenylalanine Lysine Arginine
69.3 27.4 15.5 45 19.0 50.9 63.2 38.0 30.9 52.5 18.8 46.5 16.4
f f f
0.7 0.6 0.8
f f f f f f f f f
0.6 0.4 0.7 0.6 1.6 3.1 0.2 0.7 0.2
0.1
8.9 I 0.4 9.0 f 0.1 5.4 2.3 9.0 f 0.4 (5Oyn active, 11.5 zk 0.1) 70.5 27.2 15.8 45 19.0 50.6 63.2 38.1 31.4 52.7 18.6 46.3 16.3
f f *
1.1 0.2 0.2
f f f f l f & f f
0.2 0.5 1.0 0.2 0.9 2.4 0.3 0.3 0.3
u Analysis expressed as residues per 60,000 IM, since enayme is a tetramer of identical subunits. * Four analyses of acid hydrolysates, and two analyses of base hydrolyses of samples retaining 12-20% residual activity. c Spectrophot,ometric assay of 28% active sample.
424
MACKENZIE,
Moles Sulfhydryl
STRAUS,
5
60
40
Moles Histidines
Lost/Tetramer
FIG. 4. Correlation of photoinactivaion with loss of histidines. Conditions were as in Fig. 3 and histidine was determined after acid hydrolysis. Symbols represent different experiments.
Fig. 4. About nine histidine residues per mole or two histidines per subunit are destroyed in the inactivation process. The loss of linearity at less than 20 % activity can be attributed to the fact that at this point the enzyme begins to dissociate and more histidines are available for photooxidation. Sedimentation velocity analysis showed that at 50 % inactivation, the enzyme exists completely as a tetramer, but that at 80% some dissociation to lower molecular weight species could be detected. Loss of enzymic activity upon photoinactivation could be due to loss of catalytic
in
RABINOWITZ
Lostfletramer
FIG. 3. Loss of sulfhydryl groups during photoinactivation. Conditions were 1.25 X 1e6 M enzyme, 2.5 X 10-G M methylene blue in 50 rnM Tris chloride buffer (pH 7.8) containing 50 mM KCl. Sulfhydryl determination was as described in Materials and Methods, and symbols represent different experiments.
0 .2 I2
AND
FIG. 5. Binding of ATP to formyltetrahydrofolate synthetase from C. acidi-urici. Data is presented as a Scatchard plot where r represents moles of ATP bound per mole of enzyme and r/ATP represents lO+ X r divided by the molar concentration of ATP. A. Enzyme with 47% remaining activity. Protein concentration, 8 X 10-S M; Ko = 3.2 X 1O-6 M. B. Control, fully active enzyme. Protein concentration, 8.3 X 10-z M; KD = 4 X lo+ M.
groups, the inability to bind one of the substrates, or a more general loss in conformational integrity. The ability of the enzyme to bind the substrates ATP and tetrahydropteroyltriglutate was tested in an effort to further define the cause of inactivation. Figure 5 shows that at approximately half activity, the ability of the enzyme to bind ATP was unaffected both with respect to KD and the number of ligands bound. Although the enzyme shows some loss of ability to bind tetrahydropteroyltriglutamate as shown in Fig. 6 and Table II, this does not correlate with loss in activity. No decrease in the number of tetrahydropteroyltriglutamate binding sites occurs until about 40 % inactivation. Enzyme retaining 57-32% residual activity shows binding ability decreased by approximately one site per mole enzyme. DISCUSSION
Various kinetic investigations and the study of exchange reactions have not resulted in defining the mode of action of formyltetrahydrofolate synthetase and few experiments involving chemical modification as a means to understand the mechanism
FORMYLTETRAHYDROFOLATE
425
SYNTHETASE
TABLE II SUMMARY OF LIGAND BINDING TO SYNTHETASE ~_ Per cent Moles tetrahydrofolate KD (PM) activity Per mole enzyme 100
OI
73 67 63 61 57 45 32
r
FIG. 6. Binding of tetrahydropteroyltriglutamate to formyltetrahydrofolate aynthetase from C. acidi-urici. Data is presented as a Scatchard plot where r represents moles of ligand bound per mole of enzyme and r/tetrahydropteroyltriglutamate represents 10-z X r divided by the molar concentration of ligand. Protein concentration varied from 6.8 X 10-e M to 7.4 X IF6 M in separate experiments. Enzyme had 100% (a), 67% (O), and 45$& (A) activity.
have been reported. A recent investigation (21) demonstrated that the sulfhydryl groups of formyltetrahydrofolate synthetase from C. cylindrosporum are probably not directly involved in the catalytic activity of the enzyme. It was found, however, that sulfhydryl reagents such as CTNB inactivate this synthetase and that all sulfhydryl groups are titratable in the absence of denaturants. Our preliminary experiments with the enzyme from C. acidi-urici revealed that none of the 24 sulfhydryls of this enzyme were titratable with DTNB in the absence of denaturants.4 Therefore, the synthetase from C. acidi-urici was chosen for these experiments in order to reduce t.he chances of modifying sulfhydryl groups. Photooxidation using dyes can result in extensive modification, and the residues affected can include methionine, cysteine (and cystine), histidine, tyrosine and tryptophan (15). Selectivity is obtained due to different environments of particular residues 4 Unpublished J. C. Rabinowitz.
results
of R. E. MacKenzie
and
4.1 3.6 4.1 4.2 3.8 3.6 3.8 4.1 4.1 3.1 3.2 2.7 2.9
f f * f f f f f f f f f f
0.2 0.2 0.3 0.1 0.2 0.2 0.2 0.3 0.2 0.1 0.2 0.1 0.3
2.3 1.8 2.5 2.0 2.2 2.1 2.4 2.9 2.7 1.9 2.5 2.3 3.1
3.5 3.7 3.6 3.6
f f f f
0.1 0.2 0.5 0.2
3.4 4.0 3.9 3.2
Per cent activity 100 62 47
as well as by the pH of the solution (20). In acidic media, methionine, cysteine and tryptophan are susceptible to oxidation, and histidine becomes sensitive at neutral pH. Tyrosine is destroyed most readily in alkaline solution. Under the conditions employed in this study, only histidine, methionine and cysteine residues of the synthetase were susceptible to photooxidation. However, it is possible to establish that histidine is the primary target of photooxidation by methylene blue since it is the only residue destroyed during the first 50 % inactivation. This conclusion is further substantiated by the distinctive pH rate profile and the correlation of loss of activity with the destruction of histidine residues. Cysteine and methionine are oxidized secondarily since up to 50% inactivation can be obtained before any loss of these residues occurs. The loss of enzymic activity upon photooxidation cannot be explained by the inability of the enzyme to bind the substrates ATP and tetrahydropteroyltriglutamate. No decrease in ability to bind ATP was observed with enzymes that had been
426
MACKENZIE,
STKAUS?
photooxidized to half the original activity. Although some effects on tetrahydropteroyltriglutamate binding are observed at levels of inactivation greater than 40%, it is significant that in the region of inactivation where only histidine residues are destroyed, no change in either the Ko or the number of tetrahydropteroyltriglutamatc binding sites occurs. Even beyond this level of inactivation and as low as 32% residual activity, binding affinity is relatively constant and only the number of binding sites is decreased by approximately one site per mole of cnzyme. Unlike ATP, tetrahydropteroyltriglutamate does not bind to monomeric subunits (10) and it can be expected thnrefore that the binding of tetrahydropteroyltriglutamate is more sensitive to conformational changes in the enzyme than is ATP. It is possible that with increased photooxidation a more general loss of conformation occurs. The loss of sulfhydryl groups, photooxidation of methionine and decrease in the number of tetrahydropteroyltriglutamate binding sites, all of which begin at about 50 % inactivation, lend support to this possibility. Although binding of formate cannot be determined due to its high dissociation constant (7), the results with ATP and tetrahydroptcroyltriglutamate indicate that the destruction of histidine during photoinactivation does not primarily abolish substrate binding and therefore requires that another role be assigned to this residue Additional possibilities can bc considered to explain the role of the histidine residues in this enzyme. A slight shift in positioning of substrates on the oxidized enzyme that did not permit catalysis, but at the same time did not significantly affect binding affinities, would be consistent with the results. Although this possibility cannot be ruled out. it seems unlikely that there would be no change in substrate binding affinity as was observed. The most probable explanation of the data presented, particularly during the initial 50% loss of activity, is that at least one histidine is involved in the catalytic activity of that synthrtasc:. The involvement of a second histidine is not clear, although loss of act,ivity correlates with the destruc-
AND
RABINOWITZ
tion of two histidinc>s per subunit. It is possibln that both histidines are involved or that the second residue is not important to tither binding or cat,alysis and in fact need not, be near the active site. If involved in catalysis, the histidine residue would be expected to act) as a general acid-base catalyst if the mechanism of the cnzymic reaction is concerted as suggested earlier (1). However, since enzyme bound covalent intermediates have not been unambiguously ruled out with this enzyme, a phosphoryl histidine could be involved as has been shown with succinyl CoA synthetase (22). Further, more specific chemical elucidation of the histidine residue involved is necessary to corroborate its involvement and assist in determining its mode of action. REFERENCES 1. HIMES, R. H., AND R~BINOWITZ, J. C. (1962) J. Biol. Chem. 237, 2915. 2. UYEDA, K., AND R~BINOWITZ, J. C. (1964) Arch. Biochem. Biophys. 107, 419. 3. JOYCE, B. K., AND HIMES, R. H. (1966) J. Biol. Chem. MI, 5725. 4. JOYCE, B. K., AND HIMES, R. H. (1966) J. Biol. Chem. 241, 5716. 5. CURTHOYS, N. P., AND RABINOWITZ, J. C. (1971) J. Biol. Ch.em. 246, 6942. G. MACKENZIE, R. E., AND R~\BINOWITZ, J. C. (1971) J. Biol. Chem. 246, 3731. 7. CURTHOYS, N. P., AND RABINO~ITZ, J. C. (1972) J. Biol. Chem., 247, 1965. 8. SCOTT, J. M., AND RABINOWITZ, J. C. (1967) Biochem. Biophys. Res. Commun. 29, 418. 9. WELCH, W. H., IRWIN, C. L., AND HIMES, R. H. (1968) Biochem. Biophys. Res. Commun. 30, 255. 10. CURTHOYS, N. P., STR~US, L. D., AND R~BINOWITZ, J. C. (1972) Biochemistry, 11, 345. 11. RABINO~ITZ, J. C., .~ND PRICER, W. E., JR. (19G2) J. Biol. Chem. 237, 2898. 12. BL~KELY, R. L. (1957) Biochem. J. 66, 331. 13. SAMUEL, C. E., D’ARI, L., AND RABINOWITZ, J. C. (1970) J. Biol. Chem. 246, 5115. 14. CURTHOYS, N. P., SCOTT, J. M. AND RABINOWITZ, J. C. (1972) J. Biol. Chem., 247, 1959. 15. RAY, W. J., JR. (1967) in Methods in Enzymology (C. H. W. Him, ed.), Vol. XI, p. 490, Academic Press, New York. 16. BENCZE, W. L., BND SCHMID, K. (1957) Anal. Ch.em. 29, 1193. 17. NEUMAN, N. P. (1967) in Methods in Enzymol-
FORMYLTETRAHYDROFOLATE ogy (C. H. W. Him, ed.), Vol. XI, p. 487, Academic Press, New York. 18. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70. 19. HIMES, R. H., AND WILDER, T. (1968) Arch. Biochem. Biophys. 124, 230.
SYNTHETASE
427
20. COHEN, L. A. (1968) Annu. Rev. Biochem. 37, 695. 21. NOWAK, T., AND HIMES, R. H. (1971) J. Biol. Chem. 246, 1285. 22. BRIDGER, W. A., MILLEN, W. A., AND BOYER, P. D. (1968) Biochemistry 7, 364l8.
OF
BIOCHEMISTRY
AND
Photooxidation
BIOPHYSICS
E. MACKENZIE; Department
421-427
(1972)
of Formyltetrahydrofolate Presence
ROBERT
160,
LINDA
of Biochemistry,
Synthetase
of Methylene D’ARI
University
in the
Blue’
STRAUS,
AND
JESSE C. RABINOWITZ
of CaKforrGa, Berkeley,
California
94720
Received February 2, 1972; accepted March 3, 1972 Photooxidation of formyltetrahydrofolate synthetase from Clostridium acidi-urici in the presence of methylene blue results in a pseudo-first-order loss of enzymic activity and destruction of histidine residues. In addition, a loss of cysteine and methionine residues occurs at levels of inactivation greater than 50%. Both the pH rate profle for photoinactivation which shows an inflection point near pH 6.3, as well as the correlation of loss of activity with the loss of two histidines per subunit, indicate that the primary event responsible for inactivation is the destruction of histidine. Measurements of the ability of the photooxidized enzyme to bind substrates using the method of equilibrium partitioning indicate that the loss in activity is not due to an inability to bind ATP or tetrahydropteroyl triglutamate, and thus a possible catalytic role for histidine is suggested.
The enzyme thetase (EC
cylindrosporum
formyltetrahydrofolate
syn-
6.3.4.3) from Clostridium and Clostridium acidi-urici
has been extensively investigated, but its mechanism of action is not understood. Early studies of partial exchange reactions (1) indicated that the enzyme operates by a concerted mechanism and subsequent kinetic
analyses (2, 3) and measurements of equilibrium exchange rates (4) have been consistent with that proposal, although mechanisms involving enzyme bound covalent intermediates cannot be ruled out (5). Recent
studies have shown that the enzyme
is composed of four apparently identical subunits by physical-chemical criteria (6) and that four nucleotides and four moles of tetrahydropteroyl triglutamate bind per mole of enzyme (5, 7). The tetrameric enzyme can be dissociated in the absence of certain monovalent cations to inactive
monomers (8, 9) that bind nucleotides but not tetrahydropteroyl triglutamate (10). However, the types of amino acid residues involved in substrate binding, catalysis, and the cation-dependent subunit interaction that results in formation of the tetrahydropteroyl triglutamate binding site are unknown. This investigation was undertaken to determine the nature of amino acid residues required for activity of the enzyme as well as to attempt to elucidate their role. Photooxidation of formyltetrahydrofolate synthetase from 6. acidi-urici with methylene blue results in a rapid loss of enzymic activity that correlates with the destruction of two histidine residues per subunit. MATERIALS
a The 421
@ 1972 by Academic
Press,
Inc.
METHODS
Materials. IO-Formyltetrahydrofolate synthetase was purified from C. acidi-urici by a modification of a procedure previously described (11). The modifications include chromatography on BioGel and DEAE-cellulose columns and the procedure yields a crystalline enzyme with a specific activity of 360-380 units per mg. The enzymic activity was assayed as previously described (11) .3 The sodium salt of ATP was a prod-
1 This work was supported in part by Grant AM-2109 from the National Institutes of Health, United States Public Health Service. 2 Present address: Department of Biochemistry, McGill University, Montreal 110, Quebec, Canada.
Copyright
AND
concentration
of MgClz
used is 0.1
M.
422
MACKENZIE.
STRAUS.
uct of Sigma. dl-Tetrahydrofolic acid was prepared by catalytic reducion of folic acid in aqueous solution (12) followed by purification on DEAE-cellulose (13). [aH]adenosine (G)d’-triphosphate (7.93 Ci/mmole) in 50y0 ethanol was obtained from New England Nuclear and was lyophilieed before use. Pteroyltriglutamate was a generous gift of Dr. L. Ellenbogen of Lederle Laboratories and was used in the preparation of Z-[6,7-aH2]tetrahydrofolate as described by Curthoys, Scott and Rabinowits (14) using [3H]sodium borohydride (590 mCi/mmole) from Amersham Searle. Methylene blue was a product of the National Aniline Division, Allied Chemical Corporation and ultra pure guanidine hydrochloride was purchased from Mann. Omnifluor was obtained from New England Nuclear Corp., and BiosolvBBS-3 from Beckman. All other chemicals were reagent grade. Photoinactivation. Crystals of enzyme were collected from ammonium sulfate suspensions by centrifugation, dissolved in the appropriate amount of 50 mM Tris chloride buffer (pH 7.8) containing 50 rnM KCl, and dialyzed overnight against three 500 ml changes of the same buffer. Protein concentration was determined using E%o = 8.88 X lo4 cm2/mmole (5). Photooxidation was carried out with an apparatus described by Ray (15) using flasks with optical glass bottoms for large volumes (l-2 ml) of enzyme for subsequent use in binding studies. Smaller volumes of 300 ~1 were photooxidized in 12 X 100 mm Pyrex test tubes. The temperature was maintained at 20” and the tubes were shaken by hand at one minute intervals. In kinetic experiments, 10 ~1 aliquots were removed at various times and diluted into 10 ml of 50 InM Tris chloride buffer (pH 7.8) containing 50 IIIM KCl. Aliquots (1Orrli of this diluted enzyme were assayed for activity (11). Solutions of methylene blue were made on a weight basis, and due to the instability of this dye, the concentrations quoted represent maximal values. Amino acid analysis. Samples of photoinactivated enzyme were dialyzed overnight against 1 liter of 10 mM potassium phosphate (pH 7.8) in the dark. Acid hydrolysis of the samples was carried out in evacuated tubes with 6 N HCI for 24 hr at 110”. Tryptophan was determined spectrophotometrically (16) and methionine after alkaline hydrolysis (17) in 12 X 75 mm Nalgene tubes sealed with serum caps. The tubes were evacuated and flushed with nitrogen several times before hydrolysis at 110” for 16 hr. Analyses This value was inadvertently original description.
omitted
from
the
AND
RABINOWITZ
were carried out with t,he Beckman Model 117 Automatic Amino Acid Analyzer by the singlecolumn method. Amino acid compositions were calculated relative to glutamic acid in the case of acid hydrolysis or leucine with alkaline hydrolysis. Cysteine was measured using 5,5’-dithiobis-(2nitrobenzoic acid) (18) in 6 M guanidine hydrochloride containing 1 mM EDTA and 0.008% DTNB. Absorbance at 412 nm was measured immediately after mixing. Since methylene blue did not interfere with the determination of sulfhydryl groups, the dialysis step was eliminated in this case. However, the preparation of samples was carried out in a darkened room to minimize further photooxidation. Binding of ligands. The binding of ligands as measured by two-phase partitioning w&s carried out as described by Curthoys and Rabinowitz (5, 7). Methylene blue was found not to interfere and was not removed from samples. Enzyme was used directly following photooxidation for tetrahydropteroyltriglutamate binding experiments, but was concentrated approximately IO-fold by vacuum dialysis prior to use by measuring ATP binding. Although tetrahydrofolic acid is regularly used in assaying the enzymic activity, the natural substrate, I-tetrahydropteroyltriglutamate, has greater affinity for the enzyme (7) and consequently was preferred for binding experiments. Radioactivity was measured by use of a toluene, Omnifluor, and Biosolv-BBS-3 scintillation solutions (5, 7). RESULTS
The pseudo-first-order rates of photoinactivation of formyltetrahydrofolate synthetase at three concentrations of methylene blue are shown in the semilogarithmic plot in Fig. 1. Over the range of about 0.84 moles of methylene blue per mole of enzyme, the rate of inactivation was linearly dependent upon the dye concentration. A ratio of 2 moles of methylene blue per mole of enzyme resulted in a convenient rate and was generally used in the remainder of the experiments reported here. ATP did not affect the rate of inactivation and tetrahydrofolic acid could not be tested due to its own susceptibility of oxidation. The pH rate profile for the photoinactivation process is shown in Fig. 2. Although the enzyme is less stable at pH values below 6 (19)) no loss in enzymic activity was observed during the time necessary to obtain initial rates of photoinactivation. No photo-
FORMYLTETRAHYDROFOLATE
Minutes
FIG. 1. Pseudo-first-order rate of photoinactivation of formyltetrahydrofolate synthetase. Protein was 3 mg/ml (1.25 X 1W6 M) in 50 mM Tris chloride buffer (pH 7.8) containing 50 mM KCl. Methylene blue was 1 X 1W6 M (0). 2.5 X W5 M (0) and 5 X 1W M (A).
about 20% of its original activity, followed by amino acid analysis, showed that of the photooxidizable residues (20) only histidine, methionine and a small amount of cysteine are destroyed as shown in Table I. Complete amino acid analysis showed no differences in the amounts of other amino acids. Since analysis of acid hydrolysates showed that tyrosine was not destroyed, a value of 9 residues per subunit was used in calculating the tryptophan content (16). Figure 3 and Table I show that no significant loss of sulfhydryl groups or methionine occurs until about 50% inactivation, which clearly eliminates these residues as primary targets of the photooxidation. However, a linear correlation was found between loss of activity and histidine destruction as shown TABLE
* . rate or pnotoinactivation. Final concentrations were 1.25 X lo+ M enayme, 2.5 X lO+ M methylene blue in 0.25 M potassium succinate buffers. The pH was adjusted immediately prior to inactivation. Initial rates were obtained from semilogarithmic plots of activity versus time.
inactivation was observed below pH 5.5 and a maximal rate was obtained above pH 7. The shape of this curve with an inflection point at pH 6.3 implicates the destruction of histidine as the event responsible for loss of enzymic activity. Photoinactivation of the synthetase to
I
COMPOSITION OF FORMYLTETRAHYDROFOLATESYNTHETASE AFTER PHOTOXIDATION WITH METHYLENE BLUE
AMINO
ACID
Amino acid
FIG. 2. Effect of pH on the initial
423
SYNTHETASE
Residues per monomeric unit’ Control Photooxidized*
photosensitive Histidine Tyrosine Cysteine Tryptophan” Methionine
11.3 f 9.2 f 6.1 2.3 11.2 f
0.1 0.1
photostable Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Isoleucine Leucine Phenylalanine Lysine Arginine
69.3 27.4 15.5 45 19.0 50.9 63.2 38.0 30.9 52.5 18.8 46.5 16.4
f f f
0.7 0.6 0.8
f f f f f f f f f
0.6 0.4 0.7 0.6 1.6 3.1 0.2 0.7 0.2
0.1
8.9 I 0.4 9.0 f 0.1 5.4 2.3 9.0 f 0.4 (5Oyn active, 11.5 zk 0.1) 70.5 27.2 15.8 45 19.0 50.6 63.2 38.1 31.4 52.7 18.6 46.3 16.3
f f *
1.1 0.2 0.2
f f f f l f & f f
0.2 0.5 1.0 0.2 0.9 2.4 0.3 0.3 0.3
u Analysis expressed as residues per 60,000 IM, since enayme is a tetramer of identical subunits. * Four analyses of acid hydrolysates, and two analyses of base hydrolyses of samples retaining 12-20% residual activity. c Spectrophot,ometric assay of 28% active sample.
424
MACKENZIE,
Moles Sulfhydryl
STRAUS,
5
60
40
Moles Histidines
Lost/Tetramer
FIG. 4. Correlation of photoinactivaion with loss of histidines. Conditions were as in Fig. 3 and histidine was determined after acid hydrolysis. Symbols represent different experiments.
Fig. 4. About nine histidine residues per mole or two histidines per subunit are destroyed in the inactivation process. The loss of linearity at less than 20 % activity can be attributed to the fact that at this point the enzyme begins to dissociate and more histidines are available for photooxidation. Sedimentation velocity analysis showed that at 50 % inactivation, the enzyme exists completely as a tetramer, but that at 80% some dissociation to lower molecular weight species could be detected. Loss of enzymic activity upon photoinactivation could be due to loss of catalytic
in
RABINOWITZ
Lostfletramer
FIG. 3. Loss of sulfhydryl groups during photoinactivation. Conditions were 1.25 X 1e6 M enzyme, 2.5 X 10-G M methylene blue in 50 rnM Tris chloride buffer (pH 7.8) containing 50 mM KCl. Sulfhydryl determination was as described in Materials and Methods, and symbols represent different experiments.
0 .2 I2
AND
FIG. 5. Binding of ATP to formyltetrahydrofolate synthetase from C. acidi-urici. Data is presented as a Scatchard plot where r represents moles of ATP bound per mole of enzyme and r/ATP represents lO+ X r divided by the molar concentration of ATP. A. Enzyme with 47% remaining activity. Protein concentration, 8 X 10-S M; Ko = 3.2 X 1O-6 M. B. Control, fully active enzyme. Protein concentration, 8.3 X 10-z M; KD = 4 X lo+ M.
groups, the inability to bind one of the substrates, or a more general loss in conformational integrity. The ability of the enzyme to bind the substrates ATP and tetrahydropteroyltriglutate was tested in an effort to further define the cause of inactivation. Figure 5 shows that at approximately half activity, the ability of the enzyme to bind ATP was unaffected both with respect to KD and the number of ligands bound. Although the enzyme shows some loss of ability to bind tetrahydropteroyltriglutamate as shown in Fig. 6 and Table II, this does not correlate with loss in activity. No decrease in the number of tetrahydropteroyltriglutamate binding sites occurs until about 40 % inactivation. Enzyme retaining 57-32% residual activity shows binding ability decreased by approximately one site per mole enzyme. DISCUSSION
Various kinetic investigations and the study of exchange reactions have not resulted in defining the mode of action of formyltetrahydrofolate synthetase and few experiments involving chemical modification as a means to understand the mechanism
FORMYLTETRAHYDROFOLATE
425
SYNTHETASE
TABLE II SUMMARY OF LIGAND BINDING TO SYNTHETASE ~_ Per cent Moles tetrahydrofolate KD (PM) activity Per mole enzyme 100
OI
73 67 63 61 57 45 32
r
FIG. 6. Binding of tetrahydropteroyltriglutamate to formyltetrahydrofolate aynthetase from C. acidi-urici. Data is presented as a Scatchard plot where r represents moles of ligand bound per mole of enzyme and r/tetrahydropteroyltriglutamate represents 10-z X r divided by the molar concentration of ligand. Protein concentration varied from 6.8 X 10-e M to 7.4 X IF6 M in separate experiments. Enzyme had 100% (a), 67% (O), and 45$& (A) activity.
have been reported. A recent investigation (21) demonstrated that the sulfhydryl groups of formyltetrahydrofolate synthetase from C. cylindrosporum are probably not directly involved in the catalytic activity of the enzyme. It was found, however, that sulfhydryl reagents such as CTNB inactivate this synthetase and that all sulfhydryl groups are titratable in the absence of denaturants. Our preliminary experiments with the enzyme from C. acidi-urici revealed that none of the 24 sulfhydryls of this enzyme were titratable with DTNB in the absence of denaturants.4 Therefore, the synthetase from C. acidi-urici was chosen for these experiments in order to reduce t.he chances of modifying sulfhydryl groups. Photooxidation using dyes can result in extensive modification, and the residues affected can include methionine, cysteine (and cystine), histidine, tyrosine and tryptophan (15). Selectivity is obtained due to different environments of particular residues 4 Unpublished J. C. Rabinowitz.
results
of R. E. MacKenzie
and
4.1 3.6 4.1 4.2 3.8 3.6 3.8 4.1 4.1 3.1 3.2 2.7 2.9
f f * f f f f f f f f f f
0.2 0.2 0.3 0.1 0.2 0.2 0.2 0.3 0.2 0.1 0.2 0.1 0.3
2.3 1.8 2.5 2.0 2.2 2.1 2.4 2.9 2.7 1.9 2.5 2.3 3.1
3.5 3.7 3.6 3.6
f f f f
0.1 0.2 0.5 0.2
3.4 4.0 3.9 3.2
Per cent activity 100 62 47
as well as by the pH of the solution (20). In acidic media, methionine, cysteine and tryptophan are susceptible to oxidation, and histidine becomes sensitive at neutral pH. Tyrosine is destroyed most readily in alkaline solution. Under the conditions employed in this study, only histidine, methionine and cysteine residues of the synthetase were susceptible to photooxidation. However, it is possible to establish that histidine is the primary target of photooxidation by methylene blue since it is the only residue destroyed during the first 50 % inactivation. This conclusion is further substantiated by the distinctive pH rate profile and the correlation of loss of activity with the destruction of histidine residues. Cysteine and methionine are oxidized secondarily since up to 50% inactivation can be obtained before any loss of these residues occurs. The loss of enzymic activity upon photooxidation cannot be explained by the inability of the enzyme to bind the substrates ATP and tetrahydropteroyltriglutamate. No decrease in ability to bind ATP was observed with enzymes that had been
426
MACKENZIE,
STKAUS?
photooxidized to half the original activity. Although some effects on tetrahydropteroyltriglutamate binding are observed at levels of inactivation greater than 40%, it is significant that in the region of inactivation where only histidine residues are destroyed, no change in either the Ko or the number of tetrahydropteroyltriglutamatc binding sites occurs. Even beyond this level of inactivation and as low as 32% residual activity, binding affinity is relatively constant and only the number of binding sites is decreased by approximately one site per mole of cnzyme. Unlike ATP, tetrahydropteroyltriglutamate does not bind to monomeric subunits (10) and it can be expected thnrefore that the binding of tetrahydropteroyltriglutamate is more sensitive to conformational changes in the enzyme than is ATP. It is possible that with increased photooxidation a more general loss of conformation occurs. The loss of sulfhydryl groups, photooxidation of methionine and decrease in the number of tetrahydropteroyltriglutamate binding sites, all of which begin at about 50 % inactivation, lend support to this possibility. Although binding of formate cannot be determined due to its high dissociation constant (7), the results with ATP and tetrahydroptcroyltriglutamate indicate that the destruction of histidine during photoinactivation does not primarily abolish substrate binding and therefore requires that another role be assigned to this residue Additional possibilities can bc considered to explain the role of the histidine residues in this enzyme. A slight shift in positioning of substrates on the oxidized enzyme that did not permit catalysis, but at the same time did not significantly affect binding affinities, would be consistent with the results. Although this possibility cannot be ruled out. it seems unlikely that there would be no change in substrate binding affinity as was observed. The most probable explanation of the data presented, particularly during the initial 50% loss of activity, is that at least one histidine is involved in the catalytic activity of that synthrtasc:. The involvement of a second histidine is not clear, although loss of act,ivity correlates with the destruc-
AND
RABINOWITZ
tion of two histidinc>s per subunit. It is possibln that both histidines are involved or that the second residue is not important to tither binding or cat,alysis and in fact need not, be near the active site. If involved in catalysis, the histidine residue would be expected to act) as a general acid-base catalyst if the mechanism of the cnzymic reaction is concerted as suggested earlier (1). However, since enzyme bound covalent intermediates have not been unambiguously ruled out with this enzyme, a phosphoryl histidine could be involved as has been shown with succinyl CoA synthetase (22). Further, more specific chemical elucidation of the histidine residue involved is necessary to corroborate its involvement and assist in determining its mode of action. REFERENCES 1. HIMES, R. H., AND R~BINOWITZ, J. C. (1962) J. Biol. Chem. 237, 2915. 2. UYEDA, K., AND R~BINOWITZ, J. C. (1964) Arch. Biochem. Biophys. 107, 419. 3. JOYCE, B. K., AND HIMES, R. H. (1966) J. Biol. Chem. MI, 5725. 4. JOYCE, B. K., AND HIMES, R. H. (1966) J. Biol. Chem. 241, 5716. 5. CURTHOYS, N. P., AND RABINOWITZ, J. C. (1971) J. Biol. Ch.em. 246, 6942. G. MACKENZIE, R. E., AND R~\BINOWITZ, J. C. (1971) J. Biol. Chem. 246, 3731. 7. CURTHOYS, N. P., AND RABINO~ITZ, J. C. (1972) J. Biol. Chem., 247, 1965. 8. SCOTT, J. M., AND RABINOWITZ, J. C. (1967) Biochem. Biophys. Res. Commun. 29, 418. 9. WELCH, W. H., IRWIN, C. L., AND HIMES, R. H. (1968) Biochem. Biophys. Res. Commun. 30, 255. 10. CURTHOYS, N. P., STR~US, L. D., AND R~BINOWITZ, J. C. (1972) Biochemistry, 11, 345. 11. RABINO~ITZ, J. C., .~ND PRICER, W. E., JR. (19G2) J. Biol. Chem. 237, 2898. 12. BL~KELY, R. L. (1957) Biochem. J. 66, 331. 13. SAMUEL, C. E., D’ARI, L., AND RABINOWITZ, J. C. (1970) J. Biol. Chem. 246, 5115. 14. CURTHOYS, N. P., SCOTT, J. M. AND RABINOWITZ, J. C. (1972) J. Biol. Chem., 247, 1959. 15. RAY, W. J., JR. (1967) in Methods in Enzymology (C. H. W. Him, ed.), Vol. XI, p. 490, Academic Press, New York. 16. BENCZE, W. L., BND SCHMID, K. (1957) Anal. Ch.em. 29, 1193. 17. NEUMAN, N. P. (1967) in Methods in Enzymol-
FORMYLTETRAHYDROFOLATE ogy (C. H. W. Him, ed.), Vol. XI, p. 487, Academic Press, New York. 18. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70. 19. HIMES, R. H., AND WILDER, T. (1968) Arch. Biochem. Biophys. 124, 230.
SYNTHETASE
427
20. COHEN, L. A. (1968) Annu. Rev. Biochem. 37, 695. 21. NOWAK, T., AND HIMES, R. H. (1971) J. Biol. Chem. 246, 1285. 22. BRIDGER, W. A., MILLEN, W. A., AND BOYER, P. D. (1968) Biochemistry 7, 364l8.