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[183] Tetrahydrofolic acid and formaldehyde
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
705
light (260 nm), the tetrahydropterin exhibits a dark spot, the dihydropterin a dark blue fluorescence, and the pterin a light blue fluorescence. 3. The oxygen stream (3-4 bubbles per second) is stopped as soon as the tetrahydropterin has completely disappeared. This generally occurs after 1 hour of oxidation. During this time the addition product (XXVIXXVII) begins to precipitate on the inside of the flask, from which it is removed only with great difficulty. Only the crystals that precipitate later remain free in suspension. 4. It is recommended that one employ the flask used for the preparation of the addition product (XXVI-XXVII), because a great part of the product remains attached on its inner surface. In water, all the material immediately dissolves. 5. When dry, the dihydropterin hydrochloride is very stable and may be stored for weeks without decomposition. In contrast, its oxidation in aqueous solution is very rapid, especially at pH 7.
[ 183]
Tetrahydrofolic Acid
By
and Formaldehyde
ROLAND G. KALLEN1
Tetrahydrofolic acid (THF) is the biologically active form of the vitamin, folic acid, and functions as a coenzyme in enzyme-catalyzed reactions involving one-carbon units at three oxidation levels. This communication is concerned with reactions at the intermediate (aldehyde) level of oxidation. Formaldehyde and glyoxalate react rapidly with THF in aqueous solution to produce imidazolidines: Ns,Nl°-methylene-THF (MTHF) and the carboxylate derivative, respectively.1~ Several review articles2~-2a and detailed reports on the preparation and characteristics of THF 3 and MTHF ~ have appeared previously. 1 Supported by grants from the National Science Foundation and the National Institutes of Health (HD-01247, CA-16,912, and GM 13,777). 1~R. G. Kallen and W. P. Jencks, J. Biol. Chem. 241, 5851 (1966). *~M. Friedkin, Ann. Rev. Biochem. 32, 185 (1963) and earlier reviews cited therein. ~bJ. C. Rabinowitz in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~ck, eds.), 2nd ed., Vol. 2, p. 185. Academic Press, New York, 1960. ~ E. L. R. Stokstad and I. Koch, Physiol. Rev. 47, 83 (1967). 2a R. L. Blakley, "The Biochemistry of Folic Acid and Related Pteridines," Wiley, New York, 1969. 3 F. M. Huennekens, C. K. Matthews, and K. G. Scrimgeour, Vol. VI [113]. 4 F. M. Huennekens, P. P. K. Ho, and K. G. Scrimgeour, Vol. VI [114].
706
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
R• L
II
~
t"
0
[183]
COOe
.
c.2
;ooo Properties of Tetrahydrofolic Acid and N ~ , N l ° - M e t h y l e n e t e t r a h y d r o f o l i c Acid Tetrahydrofolic Acid ( T H F ) TI-IF is available commercially in solution containing 1.0 M 2-mereaptoethanol (Nutritional Bioehemieal Corp.) or as the solid containing two moles of acetic acid per mole of THF (tool. wt. 565.4) 5 (General Bioehemieals, Sigma Chemical Corp.). Since 2-aminotetrahydropteridine-l-ones and to a significantly lesser extent NS-substituted derivatives such as M T H F are exceeding oxygen labile,6 the color of THF in all commercial preparations is tan. Recrystallization or an ether wash and precipitation from methanolether under anaerobic conditions~b,7 can be utilized to obtain more highly purified preparations. T H F in solution decomposes to dihydrofolate (absorption maximum of 282 nm), xanthopterin and other compounds in the presence of air (oxygen) in reactions that appear to be catalyzed by light, acid, base, and heavy metal ions. Copper and iron appear to be especially effective metal ion catalysts. 6 The additional fact that T H F is approximately isoionic and minimally soluble in the pH region from 2 to 5 has led to the selection of pH 5 to 6 as optimal for maintenance of stock THF solutions. Protection of solutions from light, the use of deionized water in conjunction with ethylenediaminetetraacetic acid (EDTA), 10-3 to 10-4M, and maintenance of anaerobic conditions, for example, by bubbling argon continuously, are strongly recommended. Although utilization of 2-mercaptoethanol (2-ME) to stabilize THF solutions4 is widespread, whenever this technique is adopted in kinetic and equilibrium studies, the final 2-ME concentration must be on the order of 10-4 M or less, which is lower than is commonly recommended, ~for the following reasons: (a) Hemithioacetals are formed from thiols and aldehydes in base-catalyzed reactions which are generally sufficiently rapid 6R. H. Himes and J. C. Rabinowitz, J. Biol. Chem. 237, 2903 (1962). oR. L. Blakley,Biochem. J. 65, 331 (1957). 7R. G. Kallen and W. P. Jencks, J. Biol. Chem. 241, 5845 (1966).
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
707
to compete successfully with imidazolidine formation for the available formaldehyde. F o r example, the equilibrium constant for hemithioacetal formation from 2 - M E and formaldehyde is 620 M -1 at 25 °, ionic strength 1.0 M, and accounts quantitatively for the inhibition of the rate of the reaction of T H F with formaldehyde at p H values greater t h a n 4. TM Significant inhibition is observed even at 2 - M E concentrations as low as 2.7 X 10-a M. (b) T H F has been reported to complex directly with thiols 8 to form an NS-substituted adduct, HOCH2CH2S-NS-THF. Since ultraviolet spectral alterations are observed in other NS-substituted T H F derivatives when compared with T H F , the failure to detect spectrophotometric evidence for the proposed complex in the course of experiments b y previous workers leaves this question unsettled. Stock solutions of T H F have been obtained and maintained b y the following procedure: Solid T H F is placed in serum bottles and sealed with tight-fitting TABLE I SUMMARY OF
pK'a
VALUES AND SPECTROSCOPIC DATA ON TETRAHYDROFOLIC ACID a
Tetrahydrofolate ~max d
e X 10-3 ~ (M -1 em-1)
Dissociable group
pK'
(nm)
Amide
10.5
290
21.6 b
297 220
29.1 31.4
290 270 215
22.8 25.4 40.8
292 267
20.6 15.4
265
20.6
N~
4.82
.~, Carboxyl (~) Carboxyl (a) N' N 1°
4.8 ¢ 3.5 ¢ 1.24 - 1.25
a See Kallen and Jencks. v Table reprinted by permission of the Journal of Biological Chemistry.
b Solutions of pH 12 are unstable. c Spectrophotometrieally inert. ~ = Molar absorptivity; X -- wavelength. s S. F. Zakrzewski, J. Biol. Chem. 241, 2957 (1966).
708
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[183]
silicone-greased stoppers. The gaseous phase is exchanged 5-10 times, and the appropriate amounts of deaerated E D T A solution (final cone. 10-3 M) and alkali are introduced with a syringe to achieve a pH of 5-6; the preparation is shaken to dissolve the T H F , the gaseous phase is exchanged another 5-10 times, and the solutions are stored frozen under slight positive pressure. Stock solutions of T H F (0.03 M) are briefly thawed and refrozen for aliquot removal for the dilute solutions, which are made fresh daily. The latter solutions are maintained at about pH 5 in the presence of 10-4 M ethylenediaminetetraacetic acid with argon bubbling continuously. Final f
i
i
40 3 ~.
30
7
c~
-
20
0
~"\\
~0
0 200
240
280
320
360
WaveJength (nm)
:FIG. 1. Spectra of tetrahydrofolic acid between H0 - 3.0 and pH 14. The lines represent regions in which spectra are independent of pH, that is, regions in which no spectrophotometrically detectable ionization is occurring. The pH was maintained with hydrochloric acid, sodium hydroxide, sodium formate, sodium acetate, and potassium phosphate buffers. For H0 values see M. A. Paul and F. A. Long, Chem. Rev. 57, 1 (1957). Reprinted by permission of the Journal of Biological Chemistry. reaction mixtures are derived from solutions through which argon also is bubbled slowly and continuously. When further precautions prove necessary, particularly in the alkaline region, modified Thunberg cells of quartz (Pyrocell) are used for the study of THF-containing reaction mixtures. Table I contains a summary of the pKta values, the spectral data b y which they were obtained, and the groups to which the pK'a values have been assigned. ~ The p H dependence of the spectra of the T H F is depicted in Fig. 1.
[183]
TETR&HYDROFOLATE
709
AND FORMALDI~HYDE
N~,Nl°-Methylenetetrahydrofolic Acid ( M T H F ) E q u i l i b r i a . The equilibrium constants for the formation of the various
formaldehyde adducts of T H F at 25 °, ionic strength 1.0 M, water activity 1.0 are given in Table II. ~,G,9 As the pH decreases into the region of the protonation of the N 5 site of T H F ( p K ' a = 4.82), the formation of M T H F becomes less favorable; at pH 4.3, 22 °, K .... ~u (apparent) is reported to be 1.3 X 104 M-~, ~° and at pH 2.3, 25°, K' ..... u is estimated to be 785 M -x from the p K ' ~ (3.21) of the NS-group in M T H F Scheme I) and K ' . . . . . u = K . . . . . u K ' , , / K ' , , . a~ THF + F
~
Kover~ = 3.2 X 104 M-1 ~
MTHF
pK~ = 4.82
Ha THFH * + F
~
P~2 = 3.21
MTHFH * K.~veraU = 785 M -t
SCHEME I K i n e t i c s . By making use of the spectral differences between T H F and M T H F (Figs. 1 and 2), the kinetics of M T H F formation have been studied over the pH range 0-12. la The pH dependence of the rate of M T H F formation over much of this range is depicted in Fig. 3, which shows a bell-shaped profile with the maximum rate observed at about pH 6. Scheme II (see footnote a, Table III) has been proposed to account for the available kinetic data on this reaction and postulates a change in rate-determining step at about pH 7 from general acid-catalyzed attack (step 1, Scheme II) in the acid region to general acid-catalyzed dehydration (step 2, Scheme II) in the alkaline region, la The Br0nsted alpha (Step 1) H
I
H
I
+
HCHO
k,, kl [Hal, k't' [HAI~. .~ k-l, k'-I [Hal, k~t [HA]
HO~H2 - - Ns
HI N1-6-
k____/
/ k~ [Hal, k~' [HAl/(Step/ 2) H~.
/H
H2
/c\ .,~lt m
fast
SCHEME I I
H
710
PTERIDINES, ANALOGS, AND PTERIN CO]ENZYMES 40
r
i
[183]
r
\ \
\
pH 11.5 5.2 2.3
50 ¥ E u 7
?
20
i//
©
i/
I0
20(3
240
280
520
360
Wovelength, m~
FIG. 2. Spectra of NS,Nl°-methylene-THF between pH 2 and 12. The lines represent regions in which spectra are independent of pH, that is, regions in which no spectrophotometrically detectable ionization is occurring except in the case of the pH 2.3 line, which is approximately equidistant from the pK'a of 3.2 and the next lower p K % The pH was maintained with hydrochloric acid, potassium hydroxide, and acetate buffers.
values for the attack step and dehydration steps are 0.20 and 0.75, respectively. The apparent rate constants are summarized in Table III. la Nucleophilic catalysis of the rate of formation of M T H F by secondary amines has also been observed} a
NS,NI°-Methylene-THF in Enzyme Systems Three enzyme-catalyzed hydroxymethylations and one enzymecatalyzed methylation have been reported to involve T H F and formaldehyde. ~2,13a-13° The reactions are: serine formation (serine hydroxymethylasel4), deoxyhydroxymethylcytidylate formation (deoxycytidylate 9 M. J. Osborn and F. M. Huennekens, J. Biol. Chem. 233, 969 (1958). 10 M. J. Osborn, P. T. Talbert, and F. M. Huennekens, J. Ant. Chem. Soc. 82, 4921 (1960). 11 R. G. Kallen, unpublished data. x~L. Schirch and M. Mason, J. Biol. Chem. 237, 2578 (1962) ; E. M. Wilson and E. E. Snell, ib/d., 237, 3171 (1962); L. Schirch and W. T. Jenkins, ibid., 239, 3797, 3801 (1964). laa y . C. Yeh and G. R. Greenberg, J. Biol. Chem. 242, 1307 (1967). 13b A. H. Alegria, F. M. Kahan, and J. Marmur, Biochemistry 7, 3179 (1968). 18oM. I. S. Lomax and G. R. Greenberg, J. Biol. Chem. 242, 109, 1302 (1967). 14 L-Serine: tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1.
TABLE
II
APPARENT EQUILIBRIUM CONSTANTS FOR REACTION OF TETRAHYDROFOLIC ACID AND FORMALDEHYDEa AT 25 °, IONIC STRENGTH 1.0 M , WATER ACTIVITY 1.0
/N,o--J
3.2 × 104M -Ib
KoveralI = [THF] [F]
785M -l K'overah =
[THFHOl [FI
~)Ns--CH20H ~ Kt
=
[THF] IF]
I/
OH
K2
=
32M-I c
OH 1 2.0 M-1 b
H
O
~
H2
) Htc N
Ko/Kt
=
IF]
H
m
103
CHOH 1
F = f o r m a l d e h y d e h y d r a t e , c T H F H ~ refers to t h e N 5 p r o t o n a t e d species.
712
PTERIDINES, ANALOGS, AND I~TERIN COENZYMES i
i
015 I
I
,
[183]
i
I
I I /I
OJO T
0.05
I •
2.0
L ,
4.0
6,0
8.0
pH
FIG. 3. Dependence on pH of the pseudo-first-order rate constants for the reaction of THF with 0.00167 M formaldehyde, ionic strength 1.0 M, and 25 ° in the presence (X) and absence (O) of 0.00267 M 2-mercaptoethanol. The rate constants are extrapolated to zero buffer concentration. The pH was maintained with hydrochloric acid, formate, acetate, phosphate, N-methylmorpholine, and triethylenediamine buffers. The lines at low (. . . . . ) and high ( - - . - - ) pH are the calculated rates of the attack and dehydration steps, respectively, and the upper solid line is the calculated rate from the steady state rate equation (see Kallen and Jencksla). Reprinted by permission of the Journal of Biological Chemistry. hydroxyinethylase15), d e o x y h y d r o x y m e t h y l u r i d y l a t e f o r m a t i o n ( d e o x y u r i dylate hydroxymethylase16), and thymidylate formation (thymidylate synthetase). T h e r e h a v e b e e n b a s i c a l l y two m e c h a n i s m s p r o p o s e d for t h e f o r m a t i o n of M a n n i c h bases, w h i c h h a v e b e e n p o s t u l a t e d to be i n t e r m e d i a t e s in t h e s e r e a c t i o n s ; t h e first i n v o l v e s SN2 d i s p l a c e m e n t s on e i t h e r N S - h y d r o x y m e t h y l T H F or M T H F TM ( P a t h s l a a n d l b , S c h e m e I I I ) a n d t h e s e c o n d i n v o l v e s 15Deoxycytidylate: tetrahydrofolate 5,10-hydroxymethyltransferase. ~ Deoxyuridylate: tetrahydrofolate 5,10-hydroxymethyltransferase. 16aL. Jaenicke, in "The Mechanism of Action of Water Soluble Vitamins," (A. V. S. Reuck and M. O'Connor, eds.), Ciba Syrup. No. 11, p. 38. Little, Brown, Boston, Massachusetts, 1961; F. M. Huennekens, H. R. Whiteley, and M. J. Osborn, J. Cell. Comp. Physiol. 54, Suppl. 1, p. 109 (1959). b R. L. Blakley, Biochcm. J. 65, 331 (1957); M. J. Osborn and F. M. Huennekens, J. Biol. Chem. 233, 969 (1958). c Formaldehyde is hydrated to the extent of 99.9% or greater in aqueous solution [J. F. Walker, "Formaldehyde," 3rd ed., ACS Monograph Ser., Rheinhold, New York, 1964; R. P. Bell and P. G. Evans, Proc. Roy. Soc. A291, 297 (1966)]. Reprinted by permission of the Journal of Biological Chemistry.
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
713
TABLE III APPARENT RATE CONSTANTS FOR REACTION OF TETRAHYDROFOLIC ACID AND FORMALDEHYDEa AT 25 °, IONIC STRENGTH 1.0 M, WATER ACTIVITY 1.0 b
kl k't ]c"1
v = kl v = k'l v = k"l
[THF][F] [THF][F][H +] [THF][F][H2P04 ~9]
90 M -1 sec-1 4.8 X 105 M -2 sec-1 2400 M -2 sec -1
k_l
v = k_l
r-./~/NsCH2OH
2.8 sec-1
L/ k'-i
v = k'_l
"--~'~/NsCH~OH [H +]
1.5 X 10 4 M -1 sec-1
L/
k"_l
v = k~_l
/~/N~CH~OH
[H2PO4 •]
75 M -t sec -1
[H +]
2.7 X 107 M -1 sec -1
[H,PO4e]
42 M -1 sec-1
L/
k'~
v = k'~
]~//N~CH~OH L/
[~NsCH~OH
k"2
v = k'~
k'l~
v = ]c'2~ [A][H +]
1.3 X 109M - l s e c -1 H
i
O
I O
HCH
F = formaldehyde hydrate H~N
I H ( T H F anion)
a Formaldehyde is hydrated to the extent of somewhat greater than 99.9% in aqueous solution (see references in footnote c, Table II). The apparent rate constants in this table have not been expressed in terms of unhydrated formaldehyde, which is in all probability the reactive speciesl~: This can be accomplished by multiplying the apparent rate constants by (1 + KH), where KH is the hydration constant. KH = 2275
IF]
(water activity 1.0)
b Reprinted by permission of the Journal of Biological Chemistry.
714
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
H--N Path lb
~
R
I-1
H
I+H ~
I"
[183]
kp Product
+ THF
R
Mannich base intermediate
R
I
~C~H
±H*
L
N/
-b
H,....,,- ® / J (
I
Scheme 1II
an elimination-addition sequence (Path 2, Scheme III). I~a,17 We favor an elimination-addition mechanism for formation of the Mannich base intermediate in the enzymatic reaction, rather than SN2 displacement. 1~ Proton exchange indicative of carbanion formation has been shown to occur with deoxycytidylate catalyzed by deoxycytidylate hydroxymethylase 13~ in the presence of all components except formaldehyde at a rate equal to that of the overall reaction. This has been interpreted to indicate that the formation of the deoxycytidylate carbanion is the ratedetermining step. Alanine, a glycine analog, has been shown to undergo proton exchange catalyzed by serine hydroxymethylase in the presence of THF, and the concomitant spectrophotometric changes have been attributed to the formation of the carbanion. 12 Thymidylate synthetase has been shown to catalyze the loss of tritium from the 5 position of tritium-labeled deoxyuridylate at a rate equal to 17 L. Jaenicke a n d E. Brode, Ann. 624, 120 (1959) ; E. B r o d e a n d L. 3aenieke, Biochem. Z. 332, 259 (1960).
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
715
the overall rate of thymidylate synthesis. Since this tritium loss requires the presence of all the reaction components including formaldehyde,1~¢ kinetic evidence for a carbanion intermediate has not yet been obtained. Among the possible interpretations of the data indicating tritium loss, the one proposed is that the rate-determining step in thymidylate synthesis is the further reaction of the Mannich base intermediate (step kp in Scheme III). For the hydroxymethylation reactions, the step following the formation of the Mannich base involves the replacement of THF by hydroxide in the Mannich base, while in the reaction catalyzed by thymidylate synthetase, a hydride or its equivalent from THF replaces THF from the Mannich base with the concomitant formation of dihydrofolate,is The nonenzymatic formation of thymine from a 5-thyminyl Mannich base in alkali has been reported. 19 As noted above, we favor elimination-addition mechanisms for such substitution reactions regenerating free coenzyme, but the activity of some serine hydroxymethylases12with substrate analogs that are a-substituted does not permit such a pathway in the case of those enzymes and requires another mechanism, for example, Sy2 displacement of THF fror_~ the Mannich base or perhaps a direct attack of the carbanion on formaldehyde itself without the formation of Mannich base intermediates.1Qa The stereospecific addition of formaldehyde to glyeine to form serine provides evidence against the possibility that formaldehyde itself is the reactive species39b The suitability of THF as a cofactor in these one-carbon transfer reactions appears to reside in the following considerations: 1. The high association constant for MTHF formation lowers the concentration of free formaldehyde, a compound whose toxic properties have led to its use as an antimicrobial agent. 2. The large markedly asymmetric molecule with many functional groups provides the possibility of satisfying rather stringent stereochemical binding requirements for the proper positioning of the formaldehyde moiety at the enzyme active site3 ° 3. As THF is a secondary amine, a Schiff base (imine) at the N-5 or N-10 sites of THF would be cationic and a more reactive electrophile at all pH values. 4. The relatively acidic N-5 may make the release of THF more facile than the release of a more basic aliphatic secondary amine from Mannich 18E. J. Pastore and M. Friedkin, J. Biol. Chem. 237, 3802 (1962). 19V. S. Gupta and F. M. Huennekens, Federation Proc. 24, 541 (1965). l~ap. M. Jordan and M. Akhtar, Bioehem. J. 116, 277 (1967). 19bJ.-F. Biellman and F. Schuber, Biochem. Biophys. Res. Commun. 27, 517 (1967). ~0It has been shown that /,L-THF is the active diastereoisomerin enzymesystems?d,a
716
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[184]
base intermediates, while the marked difference in basicity between N-10 and N-5 sites m a y make the formation of the N 5 imine from M T H F much more favorable on kinetic grounds3' 5. The redox properties of T H F are such as to make the hydrogen replacement of the coenzyme in thymidylate synthesis favorable. The report of an enzyme that catalyzes M T H F formation from T H F and formaldehyde ~3has been regarded with some skepticism in the absence of additional information regarding the enzyme-catalyzed reaction3 a,2d M T H F dehydrogenase 24 which catalyzes the NADP-linked interconversion of M T H F and NS,Nl°-methenyl-THF, is discussed elsewhere in this volume (see [172]). Acknowledgment We acknowledge permission received from the Journal of Biological Chemistry for the reproduction of several figures and tables. 2, Suggested by the results of studies of N,N'-diphenylethylenediamines and related THF model compoundsY.22 22S. J. Benkovic, personal communication (1969). 28M. J. Osborn, E. N. Vercamer, P. T. Talbert, and F. M. Huennekens, J. Am. Chem. Soc. 79, 6565 (1957). 245,10-Methylenetetrahydrofolate: NADP oxidoreductase (EC 1.5.1.5).
[ 184] The Nonenzymatic Methenyltetrahydrofolic
Hydrolysis
o f N 5 , N 1 o_
Acid and Related
Reactions 1
By DWIGHT R. ROBINSON The purpose of this article is to review some reactions of formyl derivatives of tetrahydrofolic acid ( T H F ) as an aid to the preparation and use of these compounds in enzymatic reactions. The synthesis of these compounds and their enzyme-catalyzed reactions are reviewed elsewhere in this and previous volumes of this series and are not considered here. Attention is focused on the catalysis of the hydrolysis of m T H F to form NI°-fTHF, and some aspects of other reactions which lead to interconversion of formyl derivatives of T H F are discussed. Abbreviations are NS-fTHF, mTHF, NI°-fTHF, and N~-formiminoTHF, which refer to the NS-formyl, NS,Nl°-methenyl, Nl°-formyl, and NS-formimino derivatives of tetrahydrofolic acid. Publication No. 513 of the Lovett Memorial Unit for the Study of Diseases Causing Deformities.
TETRAHYDROFOLATE AND FORMALDEHYDE
705
light (260 nm), the tetrahydropterin exhibits a dark spot, the dihydropterin a dark blue fluorescence, and the pterin a light blue fluorescence. 3. The oxygen stream (3-4 bubbles per second) is stopped as soon as the tetrahydropterin has completely disappeared. This generally occurs after 1 hour of oxidation. During this time the addition product (XXVIXXVII) begins to precipitate on the inside of the flask, from which it is removed only with great difficulty. Only the crystals that precipitate later remain free in suspension. 4. It is recommended that one employ the flask used for the preparation of the addition product (XXVI-XXVII), because a great part of the product remains attached on its inner surface. In water, all the material immediately dissolves. 5. When dry, the dihydropterin hydrochloride is very stable and may be stored for weeks without decomposition. In contrast, its oxidation in aqueous solution is very rapid, especially at pH 7.
[ 183]
Tetrahydrofolic Acid
By
and Formaldehyde
ROLAND G. KALLEN1
Tetrahydrofolic acid (THF) is the biologically active form of the vitamin, folic acid, and functions as a coenzyme in enzyme-catalyzed reactions involving one-carbon units at three oxidation levels. This communication is concerned with reactions at the intermediate (aldehyde) level of oxidation. Formaldehyde and glyoxalate react rapidly with THF in aqueous solution to produce imidazolidines: Ns,Nl°-methylene-THF (MTHF) and the carboxylate derivative, respectively.1~ Several review articles2~-2a and detailed reports on the preparation and characteristics of THF 3 and MTHF ~ have appeared previously. 1 Supported by grants from the National Science Foundation and the National Institutes of Health (HD-01247, CA-16,912, and GM 13,777). 1~R. G. Kallen and W. P. Jencks, J. Biol. Chem. 241, 5851 (1966). *~M. Friedkin, Ann. Rev. Biochem. 32, 185 (1963) and earlier reviews cited therein. ~bJ. C. Rabinowitz in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~ck, eds.), 2nd ed., Vol. 2, p. 185. Academic Press, New York, 1960. ~ E. L. R. Stokstad and I. Koch, Physiol. Rev. 47, 83 (1967). 2a R. L. Blakley, "The Biochemistry of Folic Acid and Related Pteridines," Wiley, New York, 1969. 3 F. M. Huennekens, C. K. Matthews, and K. G. Scrimgeour, Vol. VI [113]. 4 F. M. Huennekens, P. P. K. Ho, and K. G. Scrimgeour, Vol. VI [114].
706
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
R• L
II
~
t"
0
[183]
COOe
.
c.2
;ooo Properties of Tetrahydrofolic Acid and N ~ , N l ° - M e t h y l e n e t e t r a h y d r o f o l i c Acid Tetrahydrofolic Acid ( T H F ) TI-IF is available commercially in solution containing 1.0 M 2-mereaptoethanol (Nutritional Bioehemieal Corp.) or as the solid containing two moles of acetic acid per mole of THF (tool. wt. 565.4) 5 (General Bioehemieals, Sigma Chemical Corp.). Since 2-aminotetrahydropteridine-l-ones and to a significantly lesser extent NS-substituted derivatives such as M T H F are exceeding oxygen labile,6 the color of THF in all commercial preparations is tan. Recrystallization or an ether wash and precipitation from methanolether under anaerobic conditions~b,7 can be utilized to obtain more highly purified preparations. T H F in solution decomposes to dihydrofolate (absorption maximum of 282 nm), xanthopterin and other compounds in the presence of air (oxygen) in reactions that appear to be catalyzed by light, acid, base, and heavy metal ions. Copper and iron appear to be especially effective metal ion catalysts. 6 The additional fact that T H F is approximately isoionic and minimally soluble in the pH region from 2 to 5 has led to the selection of pH 5 to 6 as optimal for maintenance of stock THF solutions. Protection of solutions from light, the use of deionized water in conjunction with ethylenediaminetetraacetic acid (EDTA), 10-3 to 10-4M, and maintenance of anaerobic conditions, for example, by bubbling argon continuously, are strongly recommended. Although utilization of 2-mercaptoethanol (2-ME) to stabilize THF solutions4 is widespread, whenever this technique is adopted in kinetic and equilibrium studies, the final 2-ME concentration must be on the order of 10-4 M or less, which is lower than is commonly recommended, ~for the following reasons: (a) Hemithioacetals are formed from thiols and aldehydes in base-catalyzed reactions which are generally sufficiently rapid 6R. H. Himes and J. C. Rabinowitz, J. Biol. Chem. 237, 2903 (1962). oR. L. Blakley,Biochem. J. 65, 331 (1957). 7R. G. Kallen and W. P. Jencks, J. Biol. Chem. 241, 5845 (1966).
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
707
to compete successfully with imidazolidine formation for the available formaldehyde. F o r example, the equilibrium constant for hemithioacetal formation from 2 - M E and formaldehyde is 620 M -1 at 25 °, ionic strength 1.0 M, and accounts quantitatively for the inhibition of the rate of the reaction of T H F with formaldehyde at p H values greater t h a n 4. TM Significant inhibition is observed even at 2 - M E concentrations as low as 2.7 X 10-a M. (b) T H F has been reported to complex directly with thiols 8 to form an NS-substituted adduct, HOCH2CH2S-NS-THF. Since ultraviolet spectral alterations are observed in other NS-substituted T H F derivatives when compared with T H F , the failure to detect spectrophotometric evidence for the proposed complex in the course of experiments b y previous workers leaves this question unsettled. Stock solutions of T H F have been obtained and maintained b y the following procedure: Solid T H F is placed in serum bottles and sealed with tight-fitting TABLE I SUMMARY OF
pK'a
VALUES AND SPECTROSCOPIC DATA ON TETRAHYDROFOLIC ACID a
Tetrahydrofolate ~max d
e X 10-3 ~ (M -1 em-1)
Dissociable group
pK'
(nm)
Amide
10.5
290
21.6 b
297 220
29.1 31.4
290 270 215
22.8 25.4 40.8
292 267
20.6 15.4
265
20.6
N~
4.82
.~, Carboxyl (~) Carboxyl (a) N' N 1°
4.8 ¢ 3.5 ¢ 1.24 - 1.25
a See Kallen and Jencks. v Table reprinted by permission of the Journal of Biological Chemistry.
b Solutions of pH 12 are unstable. c Spectrophotometrieally inert. ~ = Molar absorptivity; X -- wavelength. s S. F. Zakrzewski, J. Biol. Chem. 241, 2957 (1966).
708
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[183]
silicone-greased stoppers. The gaseous phase is exchanged 5-10 times, and the appropriate amounts of deaerated E D T A solution (final cone. 10-3 M) and alkali are introduced with a syringe to achieve a pH of 5-6; the preparation is shaken to dissolve the T H F , the gaseous phase is exchanged another 5-10 times, and the solutions are stored frozen under slight positive pressure. Stock solutions of T H F (0.03 M) are briefly thawed and refrozen for aliquot removal for the dilute solutions, which are made fresh daily. The latter solutions are maintained at about pH 5 in the presence of 10-4 M ethylenediaminetetraacetic acid with argon bubbling continuously. Final f
i
i
40 3 ~.
30
7
c~
-
20
0
~"\\
~0
0 200
240
280
320
360
WaveJength (nm)
:FIG. 1. Spectra of tetrahydrofolic acid between H0 - 3.0 and pH 14. The lines represent regions in which spectra are independent of pH, that is, regions in which no spectrophotometrically detectable ionization is occurring. The pH was maintained with hydrochloric acid, sodium hydroxide, sodium formate, sodium acetate, and potassium phosphate buffers. For H0 values see M. A. Paul and F. A. Long, Chem. Rev. 57, 1 (1957). Reprinted by permission of the Journal of Biological Chemistry. reaction mixtures are derived from solutions through which argon also is bubbled slowly and continuously. When further precautions prove necessary, particularly in the alkaline region, modified Thunberg cells of quartz (Pyrocell) are used for the study of THF-containing reaction mixtures. Table I contains a summary of the pKta values, the spectral data b y which they were obtained, and the groups to which the pK'a values have been assigned. ~ The p H dependence of the spectra of the T H F is depicted in Fig. 1.
[183]
TETR&HYDROFOLATE
709
AND FORMALDI~HYDE
N~,Nl°-Methylenetetrahydrofolic Acid ( M T H F ) E q u i l i b r i a . The equilibrium constants for the formation of the various
formaldehyde adducts of T H F at 25 °, ionic strength 1.0 M, water activity 1.0 are given in Table II. ~,G,9 As the pH decreases into the region of the protonation of the N 5 site of T H F ( p K ' a = 4.82), the formation of M T H F becomes less favorable; at pH 4.3, 22 °, K .... ~u (apparent) is reported to be 1.3 X 104 M-~, ~° and at pH 2.3, 25°, K' ..... u is estimated to be 785 M -x from the p K ' ~ (3.21) of the NS-group in M T H F Scheme I) and K ' . . . . . u = K . . . . . u K ' , , / K ' , , . a~ THF + F
~
Kover~ = 3.2 X 104 M-1 ~
MTHF
pK~ = 4.82
Ha THFH * + F
~
P~2 = 3.21
MTHFH * K.~veraU = 785 M -t
SCHEME I K i n e t i c s . By making use of the spectral differences between T H F and M T H F (Figs. 1 and 2), the kinetics of M T H F formation have been studied over the pH range 0-12. la The pH dependence of the rate of M T H F formation over much of this range is depicted in Fig. 3, which shows a bell-shaped profile with the maximum rate observed at about pH 6. Scheme II (see footnote a, Table III) has been proposed to account for the available kinetic data on this reaction and postulates a change in rate-determining step at about pH 7 from general acid-catalyzed attack (step 1, Scheme II) in the acid region to general acid-catalyzed dehydration (step 2, Scheme II) in the alkaline region, la The Br0nsted alpha (Step 1) H
I
H
I
+
HCHO
k,, kl [Hal, k't' [HAI~. .~ k-l, k'-I [Hal, k~t [HA]
HO~H2 - - Ns
HI N1-6-
k____/
/ k~ [Hal, k~' [HAl/(Step/ 2) H~.
/H
H2
/c\ .,~lt m
fast
SCHEME I I
H
710
PTERIDINES, ANALOGS, AND PTERIN CO]ENZYMES 40
r
i
[183]
r
\ \
\
pH 11.5 5.2 2.3
50 ¥ E u 7
?
20
i//
©
i/
I0
20(3
240
280
520
360
Wovelength, m~
FIG. 2. Spectra of NS,Nl°-methylene-THF between pH 2 and 12. The lines represent regions in which spectra are independent of pH, that is, regions in which no spectrophotometrically detectable ionization is occurring except in the case of the pH 2.3 line, which is approximately equidistant from the pK'a of 3.2 and the next lower p K % The pH was maintained with hydrochloric acid, potassium hydroxide, and acetate buffers.
values for the attack step and dehydration steps are 0.20 and 0.75, respectively. The apparent rate constants are summarized in Table III. la Nucleophilic catalysis of the rate of formation of M T H F by secondary amines has also been observed} a
NS,NI°-Methylene-THF in Enzyme Systems Three enzyme-catalyzed hydroxymethylations and one enzymecatalyzed methylation have been reported to involve T H F and formaldehyde. ~2,13a-13° The reactions are: serine formation (serine hydroxymethylasel4), deoxyhydroxymethylcytidylate formation (deoxycytidylate 9 M. J. Osborn and F. M. Huennekens, J. Biol. Chem. 233, 969 (1958). 10 M. J. Osborn, P. T. Talbert, and F. M. Huennekens, J. Ant. Chem. Soc. 82, 4921 (1960). 11 R. G. Kallen, unpublished data. x~L. Schirch and M. Mason, J. Biol. Chem. 237, 2578 (1962) ; E. M. Wilson and E. E. Snell, ib/d., 237, 3171 (1962); L. Schirch and W. T. Jenkins, ibid., 239, 3797, 3801 (1964). laa y . C. Yeh and G. R. Greenberg, J. Biol. Chem. 242, 1307 (1967). 13b A. H. Alegria, F. M. Kahan, and J. Marmur, Biochemistry 7, 3179 (1968). 18oM. I. S. Lomax and G. R. Greenberg, J. Biol. Chem. 242, 109, 1302 (1967). 14 L-Serine: tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1.
TABLE
II
APPARENT EQUILIBRIUM CONSTANTS FOR REACTION OF TETRAHYDROFOLIC ACID AND FORMALDEHYDEa AT 25 °, IONIC STRENGTH 1.0 M , WATER ACTIVITY 1.0
/N,o--J
3.2 × 104M -Ib
KoveralI = [THF] [F]
785M -l K'overah =
[THFHOl [FI
~)Ns--CH20H ~ Kt
=
[THF] IF]
I/
OH
K2
=
32M-I c
OH 1 2.0 M-1 b
H
O
~
H2
) Htc N
Ko/Kt
=
IF]
H
m
103
CHOH 1
F = f o r m a l d e h y d e h y d r a t e , c T H F H ~ refers to t h e N 5 p r o t o n a t e d species.
712
PTERIDINES, ANALOGS, AND I~TERIN COENZYMES i
i
015 I
I
,
[183]
i
I
I I /I
OJO T
0.05
I •
2.0
L ,
4.0
6,0
8.0
pH
FIG. 3. Dependence on pH of the pseudo-first-order rate constants for the reaction of THF with 0.00167 M formaldehyde, ionic strength 1.0 M, and 25 ° in the presence (X) and absence (O) of 0.00267 M 2-mercaptoethanol. The rate constants are extrapolated to zero buffer concentration. The pH was maintained with hydrochloric acid, formate, acetate, phosphate, N-methylmorpholine, and triethylenediamine buffers. The lines at low (. . . . . ) and high ( - - . - - ) pH are the calculated rates of the attack and dehydration steps, respectively, and the upper solid line is the calculated rate from the steady state rate equation (see Kallen and Jencksla). Reprinted by permission of the Journal of Biological Chemistry. hydroxyinethylase15), d e o x y h y d r o x y m e t h y l u r i d y l a t e f o r m a t i o n ( d e o x y u r i dylate hydroxymethylase16), and thymidylate formation (thymidylate synthetase). T h e r e h a v e b e e n b a s i c a l l y two m e c h a n i s m s p r o p o s e d for t h e f o r m a t i o n of M a n n i c h bases, w h i c h h a v e b e e n p o s t u l a t e d to be i n t e r m e d i a t e s in t h e s e r e a c t i o n s ; t h e first i n v o l v e s SN2 d i s p l a c e m e n t s on e i t h e r N S - h y d r o x y m e t h y l T H F or M T H F TM ( P a t h s l a a n d l b , S c h e m e I I I ) a n d t h e s e c o n d i n v o l v e s 15Deoxycytidylate: tetrahydrofolate 5,10-hydroxymethyltransferase. ~ Deoxyuridylate: tetrahydrofolate 5,10-hydroxymethyltransferase. 16aL. Jaenicke, in "The Mechanism of Action of Water Soluble Vitamins," (A. V. S. Reuck and M. O'Connor, eds.), Ciba Syrup. No. 11, p. 38. Little, Brown, Boston, Massachusetts, 1961; F. M. Huennekens, H. R. Whiteley, and M. J. Osborn, J. Cell. Comp. Physiol. 54, Suppl. 1, p. 109 (1959). b R. L. Blakley, Biochcm. J. 65, 331 (1957); M. J. Osborn and F. M. Huennekens, J. Biol. Chem. 233, 969 (1958). c Formaldehyde is hydrated to the extent of 99.9% or greater in aqueous solution [J. F. Walker, "Formaldehyde," 3rd ed., ACS Monograph Ser., Rheinhold, New York, 1964; R. P. Bell and P. G. Evans, Proc. Roy. Soc. A291, 297 (1966)]. Reprinted by permission of the Journal of Biological Chemistry.
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
713
TABLE III APPARENT RATE CONSTANTS FOR REACTION OF TETRAHYDROFOLIC ACID AND FORMALDEHYDEa AT 25 °, IONIC STRENGTH 1.0 M, WATER ACTIVITY 1.0 b
kl k't ]c"1
v = kl v = k'l v = k"l
[THF][F] [THF][F][H +] [THF][F][H2P04 ~9]
90 M -1 sec-1 4.8 X 105 M -2 sec-1 2400 M -2 sec -1
k_l
v = k_l
r-./~/NsCH2OH
2.8 sec-1
L/ k'-i
v = k'_l
"--~'~/NsCH~OH [H +]
1.5 X 10 4 M -1 sec-1
L/
k"_l
v = k~_l
/~/N~CH~OH
[H2PO4 •]
75 M -t sec -1
[H +]
2.7 X 107 M -1 sec -1
[H,PO4e]
42 M -1 sec-1
L/
k'~
v = k'~
]~//N~CH~OH L/
[~NsCH~OH
k"2
v = k'~
k'l~
v = ]c'2~ [A][H +]
1.3 X 109M - l s e c -1 H
i
O
I O
HCH
F = formaldehyde hydrate H~N
I H ( T H F anion)
a Formaldehyde is hydrated to the extent of somewhat greater than 99.9% in aqueous solution (see references in footnote c, Table II). The apparent rate constants in this table have not been expressed in terms of unhydrated formaldehyde, which is in all probability the reactive speciesl~: This can be accomplished by multiplying the apparent rate constants by (1 + KH), where KH is the hydration constant. KH = 2275
IF]
(water activity 1.0)
b Reprinted by permission of the Journal of Biological Chemistry.
714
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
H--N Path lb
~
R
I-1
H
I+H ~
I"
[183]
kp Product
+ THF
R
Mannich base intermediate
R
I
~C~H
±H*
L
N/
-b
H,....,,- ® / J (
I
Scheme 1II
an elimination-addition sequence (Path 2, Scheme III). I~a,17 We favor an elimination-addition mechanism for formation of the Mannich base intermediate in the enzymatic reaction, rather than SN2 displacement. 1~ Proton exchange indicative of carbanion formation has been shown to occur with deoxycytidylate catalyzed by deoxycytidylate hydroxymethylase 13~ in the presence of all components except formaldehyde at a rate equal to that of the overall reaction. This has been interpreted to indicate that the formation of the deoxycytidylate carbanion is the ratedetermining step. Alanine, a glycine analog, has been shown to undergo proton exchange catalyzed by serine hydroxymethylase in the presence of THF, and the concomitant spectrophotometric changes have been attributed to the formation of the carbanion. 12 Thymidylate synthetase has been shown to catalyze the loss of tritium from the 5 position of tritium-labeled deoxyuridylate at a rate equal to 17 L. Jaenicke a n d E. Brode, Ann. 624, 120 (1959) ; E. B r o d e a n d L. 3aenieke, Biochem. Z. 332, 259 (1960).
[183]
TETRAHYDROFOLATE AND FORMALDEHYDE
715
the overall rate of thymidylate synthesis. Since this tritium loss requires the presence of all the reaction components including formaldehyde,1~¢ kinetic evidence for a carbanion intermediate has not yet been obtained. Among the possible interpretations of the data indicating tritium loss, the one proposed is that the rate-determining step in thymidylate synthesis is the further reaction of the Mannich base intermediate (step kp in Scheme III). For the hydroxymethylation reactions, the step following the formation of the Mannich base involves the replacement of THF by hydroxide in the Mannich base, while in the reaction catalyzed by thymidylate synthetase, a hydride or its equivalent from THF replaces THF from the Mannich base with the concomitant formation of dihydrofolate,is The nonenzymatic formation of thymine from a 5-thyminyl Mannich base in alkali has been reported. 19 As noted above, we favor elimination-addition mechanisms for such substitution reactions regenerating free coenzyme, but the activity of some serine hydroxymethylases12with substrate analogs that are a-substituted does not permit such a pathway in the case of those enzymes and requires another mechanism, for example, Sy2 displacement of THF fror_~ the Mannich base or perhaps a direct attack of the carbanion on formaldehyde itself without the formation of Mannich base intermediates.1Qa The stereospecific addition of formaldehyde to glyeine to form serine provides evidence against the possibility that formaldehyde itself is the reactive species39b The suitability of THF as a cofactor in these one-carbon transfer reactions appears to reside in the following considerations: 1. The high association constant for MTHF formation lowers the concentration of free formaldehyde, a compound whose toxic properties have led to its use as an antimicrobial agent. 2. The large markedly asymmetric molecule with many functional groups provides the possibility of satisfying rather stringent stereochemical binding requirements for the proper positioning of the formaldehyde moiety at the enzyme active site3 ° 3. As THF is a secondary amine, a Schiff base (imine) at the N-5 or N-10 sites of THF would be cationic and a more reactive electrophile at all pH values. 4. The relatively acidic N-5 may make the release of THF more facile than the release of a more basic aliphatic secondary amine from Mannich 18E. J. Pastore and M. Friedkin, J. Biol. Chem. 237, 3802 (1962). 19V. S. Gupta and F. M. Huennekens, Federation Proc. 24, 541 (1965). l~ap. M. Jordan and M. Akhtar, Bioehem. J. 116, 277 (1967). 19bJ.-F. Biellman and F. Schuber, Biochem. Biophys. Res. Commun. 27, 517 (1967). ~0It has been shown that /,L-THF is the active diastereoisomerin enzymesystems?d,a
716
PTERIDINES, ANALOGS, AND PTERIN COENZYMES
[184]
base intermediates, while the marked difference in basicity between N-10 and N-5 sites m a y make the formation of the N 5 imine from M T H F much more favorable on kinetic grounds3' 5. The redox properties of T H F are such as to make the hydrogen replacement of the coenzyme in thymidylate synthesis favorable. The report of an enzyme that catalyzes M T H F formation from T H F and formaldehyde ~3has been regarded with some skepticism in the absence of additional information regarding the enzyme-catalyzed reaction3 a,2d M T H F dehydrogenase 24 which catalyzes the NADP-linked interconversion of M T H F and NS,Nl°-methenyl-THF, is discussed elsewhere in this volume (see [172]). Acknowledgment We acknowledge permission received from the Journal of Biological Chemistry for the reproduction of several figures and tables. 2, Suggested by the results of studies of N,N'-diphenylethylenediamines and related THF model compoundsY.22 22S. J. Benkovic, personal communication (1969). 28M. J. Osborn, E. N. Vercamer, P. T. Talbert, and F. M. Huennekens, J. Am. Chem. Soc. 79, 6565 (1957). 245,10-Methylenetetrahydrofolate: NADP oxidoreductase (EC 1.5.1.5).
[ 184] The Nonenzymatic Methenyltetrahydrofolic
Hydrolysis
o f N 5 , N 1 o_
Acid and Related
Reactions 1
By DWIGHT R. ROBINSON The purpose of this article is to review some reactions of formyl derivatives of tetrahydrofolic acid ( T H F ) as an aid to the preparation and use of these compounds in enzymatic reactions. The synthesis of these compounds and their enzyme-catalyzed reactions are reviewed elsewhere in this and previous volumes of this series and are not considered here. Attention is focused on the catalysis of the hydrolysis of m T H F to form NI°-fTHF, and some aspects of other reactions which lead to interconversion of formyl derivatives of T H F are discussed. Abbreviations are NS-fTHF, mTHF, NI°-fTHF, and N~-formiminoTHF, which refer to the NS-formyl, NS,Nl°-methenyl, Nl°-formyl, and NS-formimino derivatives of tetrahydrofolic acid. Publication No. 513 of the Lovett Memorial Unit for the Study of Diseases Causing Deformities.