A STUDY OF THE MECHANISM OF SERINE BIOSYNTHESIS*

BY ROY L. KISLIUK AND WARWICK SAKAMI

(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland, Ohio)

(Received for publication, September 21, 1954)

Elwyn et al. (1, 2) have demonstrated that the C1 unit formed from serine is of the oxidation level of formaldehyde rather than formate. They compared the dilution of the Cl4 and deuterium of 2 ,3-deuterio-3-C14,N15- L-serine that occurred during the conversion of the serine B-carbon to methyl groups in rats. The results indicated that this carbon did not pass through an intermediate, such as formate, which required the loss of 1 of the hydrogen atoms. In consideration of the results of Elwyn et al. and the general implication of the cit,rovorum factor, 5-formyl-5,6,7 ,&tetra- hydrofolic acid, in the metabolism of C1 compounds, Welch and Nichol (3) proposed that the C1 unit formed from serine was a reduced form of citro- vorum factor, 5-hydroxymethyl-5,6,7,8-tetrahydrofolic acid, a hypothet- ical condensation product of formaldehyde and tetrahydrofolic acid.

The formulation of the biosynthesis of serine as a process involving a condensation between the a-carbon of glycine, activated by the Schiff base formation with pyridoxal phosphate (4)) and 5-hydroxymethyl-5,6,7,8- tetrahydrofolic acid is reminiscent of the Mannich reaction (5). Serine would be produced by hydrolytic cleavage of the resulting product. The conversion of serine to glycine could occur by the reverse process.

The participation of THFA’ as a cofactor in the biosynthesis and cleavage of serine has been studied by adding it to pigeon liver extracts incubated anaerobically with glycine-1-V and L-serine. The assumption underlying this procedure was that the formation and breakdown of serine were catalyzed by a single enzyme. In this case, the ,&carbon atom of the L-serine would provide the C1 unit for the conversion of glycine-l-Cl4

* The results of preliminary experiments in this investigation have been published (,I. Am. Chem. Sot., 76, 1456 (1954)).

This investigation has been supported by a grant-in-aid from the American Can- cer Society upon recommendation of the Committee on Growth of the National Research Council.

1 The following abbrevint~ions are employed in this paper: THFA = 5,6,7,8- tetrah,vdrofolic acid; DHFA = 7,%dihydrofolic acid; E’A = folic acid; TIIFA- CH,OH = 5-hydroxymethyl-5,6,7,%tetrahydrofolic acid; THFA-CHO = citrovorum factor; ATP = adenosinetriphosphate; G-6-P = glucose-6-phosphate; DPN = diphosphopyridine nucleotide; TPN = triphosphopyridine nucleotide.

47

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48 SERINE BIOSYNTHESIS

to serine-1-C4. The sole addition of tetrahydrofolic acid produced a

CHzOHCHNH&OOH F? Cl + CHzNHzCOOH

Cl + CH2NH&*OOH F? CHzOHCHNH&*OOH

pronounced stimulation of the incorporation of glycine-l-Cl4 into serine even when the extract had been treated previously with Dowex 1 chloride and dialyzed. The incorporation of formaldehyde and formate into ser- ine has also been studied, and it has been possible to reactivate treated2 extracts by the addition of THFA and, in the case of formate, of ATP and a reducing system.

EXPERIMENTAL

Materials and Methods

5,6,7, S-Tetrahydrofolic acid was prepared by the method of Broquist et al. (6)) 7, S-dihydrofolic acid by the procedure of O’Dell et al. (7)) formate- Cl4 according to Melville et al. (S), and formaldehyde-U4 by the procedure of Siegel and Lafaye (9).

Male pigeons were obtained from the Palmetto Pigeon Plant. Phosphate buffer extracts of pigeon liver were prepared by the procedure

of Berg (lo), lyophilized, and stored in vacua at -10”. Extracts were treated with Dowex 1 chloride (50 to 100 mesh, 4 per cent cross-linked) by the following procedure: 880 mg. of the lyophilized powder were dissolved in 8 ml. of ice-cold distilled water and stirred with 3 gm. of Dowex resin (prepared by the method of Chantrenne and Lipmann (11) and dried in air) in an ice bath for 10 minutes. After removal of the resin by centrif- ugation at O”, the extract was diluted with an equal volume of cold 0.2 M

potassium phosphate buffer, pH 7.5, and dialyzed in Visking NoJax casing (S/32 inches) suspended in flowing 0.1 M potassium phosphate buffer, pH 7.5, at 2’. Extracts treated with Dowex 1 chloride and dialyzed were stable toward lyophilization.

Incubation was carried out in Thunberg tubes at 34”. The radioactive substrate was placed in the side arm and all other components of the in- cubation mixture were added to the main compartment which was im- mersed in an ice bath. The gas phase was hydrogen at atmospheric pressure. At the end of the incubation 1 ml. of 20 per cent trichloroacetic acid was added and the mixture was centrifuged. The value for the “total activity fixed” (Table V) was determined by adding 0.1 ml. of the super- natant solution to 2 ml. of 1 N HCl in a Pyrex ashing dish and evaporating to dryness under an infra-red lamp. The activity of the residue was determined with an end window Geiger-Mtiller counter.3 Serine and

2 Treated with Dowes 1 chloride and dialyzed. 3 The self-absorption of these samples was determined to be negligible.

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R. L. KISLIUK AND W. SAKAMI 49

glycine were isolated from aliquots of the protein-free incubation mixture by chromatography on Dowex 50 (100 to 200 mesh, 12 per cent cross- linked) with 1 N HCl as the eluent. When radioactive glycine as well as serine was present, 50 X 1 cm. columns were used; otherwise 25 X 1 cm. columns were employed. The activity of the serine and glycine was determined by evaporating aliquots of the respective fractions on Pyrex ashing dishes and measuring radioactivity as in the determination of total “fixed” activity.3 This procedure did not cause loss of activity of the samples.

The identification of the activity of the serine fraction of the Dowex chromatogram with serine was further established by ascending paper chromatography with Whatman No. 4 filter paper and 80 per cent pyridine- water as the solvent. All of the radioactivity on the paper was located in a single spot possessing the Rp of L-serine.

Serine was determined by a modification of the procedure of Frisell et al. (12) in which formaldehyde resulting from the periodate oxidation of serine was determined with SchifYs reagent rather than with chromotropic acid. In our hands this modification permitted greater precision.

The activity of the @-carbon of serine was determined by dilution of the formaldehyde formed from serine by periodate oxidation with carrier formaldehyde and conversion to the dimedon derivative. An aliquot of an alcohol solution of the formaldehydemethone was evaporated on a Pyrex ashing dish and counted.3 Protein was determined by the biuret method 03).

Results

Pigeon liver extracts possess varying abilities to interconvert glycine and serine. This ability is slowly lost on storage of the lyophilized powder. The extract used in the experiment shown in Table I had been stored for several months and had retained very little activity. The sole addition of THFA produced a marked increase in the radioactivity of the serine. It has not been possible to demonstrate any other requirement of this reaction. When the extracts were treated with Dowex 1 chloride and subjected to prolonged dialysis, they were restored to the activity of the original prep- aration supplemented with THFA by the sole addition of THFA (Table I). The further addition of ATP, nL-homocysteine, or DPN (Table II) produced no additional activation. The incorporation of formate-Cl* in- to serine (10) and the interconversion of glycine and serine* in untreated pigeon liver extracts are stimulated by m-homocysteine. However, ho- mocysteine does not increase the incorporation of glycine into serine in treated2 extracts with or without THFA supplementation.

4 Unpublished experiments of R. L. Kisliuk and W. Sakami.

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50 SERINE BIOSYNTHESIS

Folic acid could not replace tetrahydrofolic acid in stimulating the incor- poration of glycine-Cl* into serine and was inhibitory when added together with THFA.

TABLE I

Efect of THFA on Incorporation of Glycine-l-04 into Serine

Preparation

c.pm.

1. Untreated extract 200 2. “ “ 7600 3. Treated extract 300 *. “ “ 7600 5. “ “ 0 6. “ “ 5200

1 ml. of untreated or Dowex-treated, dialyzed (15 hours) extract (30 mg. of pro- tein), 3.5rmoles of glycine-1-W (16,600 c.p.m. total), 8 pmoles of L-serine, 3.5pmoles of THFA, 3.0 @moles of FA, 100rmoles of potassium phosphate buffer, pH 7.5. Total volume 2 ml. Incubation 1 hour.

-

-

None THFA None THFA FA THFA + FA

Addition Total activity of serine

TABLE II

Effect of Various Additions on Incorporation of Glycine-i-Cl4 into Serine

Additions I Activity of serine

c.p.m.

1. None........................................... 1180 2. nn-Homocysteine............................... 1200 3. THFA. 2770 4. ‘( + ATP 2730 5. “ + nL-homocysteine...................... 2760 6. “ + DPN.. 2790 7. “ (45 min. incubation). _. .: 4310

0.5 ml. of Dowex-treated, dialyzed (22 hours) extract (14 mg. of protein), 10 flmoles of glycine-1-W (11,000 c.p.m.), 10 pmoles of L-serine, 5.0 pmoles of nn-homo- cysteine, 3.5 rmoles of THFA, 10 pmoles of ATP, 1.0 pmole of DPN, 100 rmoles of potassium phosphate buffer, pH 7.5. Total volume 2 ml. Incubation 11 minutes.

Results similar to these have been briefly reported by Blakley (14). The incorporation of formaldehyde and formate into serine was studied

by incubating pigeon liver extracts with formaldehyde-Cl4 or formate-C?* and glycine and determining the amount and the total activity of the serine formed. The addition of THFA to inactivated extracts strongly stimulated the incorporation of formaldehyde-Cl4 into serine (Tables III and IV). The specific activity of the serine and of the serine P-carbon was

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R. L. KISLIUK AND W. SAKAMI 51

determined (Table IV) and found to be equal to that of the formaldehyde used in the incubation within the experimental error. This demonstrated

TABLE III

Incorporation of Formaldehyde-C’” into Serine in Pigeon Liver Extract

Preparation Additions

Treated None “ THFA “ “ + ATP ‘I “ + nn-homocysteine “ DHFA

Untreated None “ THFA ‘I ‘I + ATP “ “ + nn-homocysteine “ DHFA

Activity in swine

c.p.ni.

180

112,000 124,000

5J,m 7,500 1,350

96,000 90,000 51 ,ooo 47,000

1 ml. of extract untreated (37 mg. of protein) or treated twice with Dowex 1 chlo- ride and dialyzed (24 hours) (25 mg. of protein), 6.5 pmoles of HCHO-Cl4 (280,000 c.p.m.), 10 pmoles of glycine, 10 rmoles of ATP, 5 pmoles of nn-homocysteine, 3.5 rmoles of THFA, 3.5 rmoles of DHFA. Total volume 2 ml. Incubation 13 minutes.

TABLE IV

Effect of Folic Acid Derivatives on Formaldehyde Utilization

Additions NOW2 FA DHFA THFA

Activity in serine

c.p.?% c.p.?tt. C2.p.m. C.).tn.

1. None................................ 110 140 3,650 67,500* 2. ATP.. . . . 110 3,850 61,000 3. “ DPN, MnSOI, G-6-P.. . . 580 54,000 61,000

1 ml. of Dowex l-treated, dialyzed (24 hours) extract (40 mg. of protein), 6.5 rmoles of formaldehyde-Cl4 (230,000 c.p.m.), 10 rmoles of glycine, 3.5 rmoles of FA, 3.5 pmoles of DHFA, 3.5 pmoles of THFA, 10 rmoles of ATP, 1.0 pmole of DPN, 1 .O pmole of MnSOI, 10 pmoles of glucose-g-phosphate, 100 pmoles of potassium phos- phate buffer, pH 7.5. Preincubation 7 minutes before tipping in formaldehyde. Total volume 2 ml. Incubation 5 minutes.

* The specific activities of the serine, the serine p-carbon isolated, and the for- maldehyde added t,o the incubat,ion mixture were 45,000,41,300, and 43,000 c.p.m. per jmole, respectively.

that the incorporation of formaldehyde activity into serine occurred by a net synthesis. As in the case of the interconversion react’ion of serine and glycine, it has not been possible to detect any other cofactor requirement of

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52 SERINE BIOSYNTHESIS

this reaction. The activity of the treated2 extract stimulated by THFA was approximately equal to that of the untreated pigeon liver preparation similarly supplemented (Table IV). Moreover, additional supplementa- tion with ATP or with a mixture of ATP, DPN, Mn++, and glucose-g- phosphate produced no further effect in either preparation. This mixture was added with the assumption that it would reconstitute the glycolytic system of pigeon liver extract, thereby increasing the supply of ATP. It is also provided reduced DPN. Homocysteine decreased the activity

TABLE V

Effect of I’ctrahydrofolic Acid and Other Substances on Incorporation of Fornzate-C~4 into Serine @-Carbon

Ex%:Yt

1

2

--

-

None THFA

‘I + ATP + DPN + MnSO, + G-6-P ATP + DPN + MnSO, + G-6-P THFA + ATP + DPN + MnSO, + G-6-P

“ less ATP “ “ DPN “ “ G-6-P I‘ “ MnSO,

Untreated extract + THFA + ATI’ + DPN + MnSO, + G-6-P

Activity of serine

c.p.m.

20

220

79,ooo* 180

98,000 ‘3,500

27,000 1,600

28,000 100,000

T

-

Total activity fixed

C.).rn.

95,000 6,300

45,000 82,000 28,500

112,000

1 ml. of Dowex l-treated, dialyzed (24 hours) extract (Experiment 1, 45 mg. of protein, Experiment 2, 41 mg. of protein) or untreated extract (45 mg. of protein), 5 amoles of formate-Cl’ (270,000 c.p.m.), 10 pmoles of glycine, 3.5 amoles of THFA, 10 pmoles of ATP, 1.0 pmole of DPN, 2.0 pmoles of MnSO,, 20 pmoles of G-6-P. Total volume 2 ml. Incubation 30 minutes.

* The specific activities of the serine, the serine B-carbon, and the formate added to the medium were 59,000,51,000, and 54,000 c.p.m. per pmole, respectively.

incorporated into serine as would be expected according to its ability to bind formaldehyde (10). DHFA was much more strongly stimulatory in the untreated than in the treated extract (Table IV). Although the ad- dition of DHFA produced very low serine activity when added to the treat.ed2 extract, further supplementation of the extract with ATP, DPN, Mn++, and glucose-6-phosphate raised the activity of the serine almost to that produced by the addition of THFA. FA was slightly active under these conditions.

Similar results have been briefly reported by Blakley (14). The addition of THFA alone to treated2 pigeon liver extracts did not

produce a significant increase in its ability to incorporate formate-Cl4 into

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R. L. KISLIUK AND W. SAKAMI 53

serine (Table V). This was not unexpected, since the conversion of for- mate to the serine &carbon involves a reductive step. The further addi- tion of ATP, DPN, MnS04, and glucose-6-phosphate produced a marked incorporation of formate carbon into serine (Table V). The specific ac- tivity of the serine and of the serine P-carbon was determined (Table V) and found to be approximately equal to that of the formate, indicating that the introduction of formate-Cl4 into serine occurred by a net synthesis. The activity of the serine was similar to that produced by untreated enzyme supplemented in the same manner (Table V).

In agreement with the report of Berg (lo), homocysteine stimulated the incorporation of formate-Cl4 into serine in the untreated extract. HOW- ever, it did not significantly activate the treated2 preparation. The effect of single omissions from the additions of ATP, DPN, MnS04, and glucose- 6-phosphate on the activity of the serine has been determined. In the absence of any one of these substances the radioactivity of the serine was diminished. The absence of DPN or glucose-6-phosphate resulted in an accumulation of formate-Cl4 in some compound that was not serine. The nature of this substance is being investigated. These results suggest that reduced DPN is not required in the initial phase of formate utilization.

DISCUSSION

The finding that the conversion of glycine-Cl4 to serine-Cl4 in the pres- ence of L-serine is strongly stimulated by the sole addition of THFA6* 6 supports the hypothesis that the formation of serine from glycine and the breakdown of serine are catalyzed by a single enzyme for which the name serine hydroxymethylase is proposed since it catalyzes the removal and addition of a hydroxymethyl group. It is uncertain whether THFA itself, or a derivative, is the physiological cofactor of this reaction. Folic acid and citrovorum factor occur in animal tissues largely in bound form (15-19) and it is possible that the cofactor of serine hydroxymethylase is a similar derivative of THFA.

The scheme relating formate, formaldehyde, and serine shown in Fig. 1 is tentatively proposed to account for the results obtained in the present investigation. This scheme is similar to the one proposed by Berg (10) except that homocysteine has been replaced by THFA.6 According to the present hypothesis, the C1 unit directly involved in serine biosynthesis is THFA-CHZOH.~ Formaldehyde is utilized for the formation of the serine P-carbon by enzymatic condensation with THFA5 producing THFA-

5 Or a derivative. 6 Serine hydroxymethylase has been purified by precipitation with ammonium

sulfate (between 25 and 40 per cent saturated at 2”). This preparation possessed 4 times the specific activity of the original and was activated by the sole addition of THFA.

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54 SERINE I3IOSTNTIIESIS

CH2OII.5 Formate utilization is assumed to occur by a pathway in which THFA5 and formate are combined in the presence of ATP and Mnff to form THFA-CH0.5 THFA-CHOb is then reduced to THFA-CH,OH’ by a DPN enzyme system. It is also possible t,hat TPS rather than l)PX is involved in this reduction. DPN is converted to TPN in liver in the presence of ATP (LO).

The enzymatic nature of the formation of THFA-CHzOH5 from THFA5 and formaldehyde has been indicated by studies of deoxycholic acid ex- t,racts of rat liver particles.’ Wh& supplemented with THFA, this prep- aration catalyzes the rapid interconversion of serine and glycine but does

Serine-B-Carbon

II

Thymine-CH3

/

Choline-CH3

THFA Formaldehyde XL Hydroxymethyl-THFA

(derivative ? )

DPNH PN

ATP lb Formate

Mn++ c Citrovorum Factor

(derivative ? ) THFA

T 11 ZDPNH Purim.9

Folic Acid

FIQ. 1. Interrelationships between l-carbon compounds

not utilize formaldehyde as would be expected if the formation of THFA- CHtOH were solely a spontaneous process.

The effect of single omissions from the mixture of ATP, DPN, Mn++, and glucose-6-phosphate is consistent with this scheme. When ATP or Mn++ was omitted, the amount of formate-Cl4 incorporated into non-acid- volatile substances was considerably reduced and all of this activity was accounted for as serine. These findings are consistent with a r61e of ATP and Mn++ in the initial process of formate utilization. When DPN or glucose-6-phosphate was omitted, considerable formate was converted to a substance that was not serine but which may have been citrovorum factor. The formation of citrovorum factor from folic acid in liver has been ob- served by Nichol (21). These results are consistent with the hypothesis that the initial step in formate utilization does not involve reduction and that formate is not converted to serine via formaldehyde.

7 Unpublished experiments of S. Deodhar and W. Sakami.

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R. L. KISLIUK AND W. SAKAMI 55

The scheme shown in Fig. 1 is consistent with the literature on the relationships of compounds possessing a single carbon atom. It is also consistent with the indication that formate and formaldehyde do not mldergo obligatory interconversion in their incorporation into carbons 2 and 8 of purine (22) but are metabolized via a common intermediate. Furthermore, the scheme agrees with tracer studies showing that the car- bons of @-deuterioserine-Cl4 and deuterioformate-Cl4 arc incorporated into choline (23, 1, 24) and thymine (1, 24) methyl groups with little or no loss of deuterium. In the case of the formate utilization, the interconversion of citrovorum factor and THFA-CH,OH would not be expected to produce loss of carbon-bound deuterium. Hydrogen would be expected to add to the formyl group in the reduction of the cit’rovorum factor, as in most biological reactions, with a single spatial distribution and would be the same hydrogen atom removed in the reversal of this process. In the case of serine, the results are consistent with the existence of a THFA-CH,OH intermediate which can undergo rapid reversible conversion to citrovorum factor though not with obligatory conversion to the latter compound. Ratios for W:D dilution of 0.97 to 0.89 (1) and of 0.75 and 0.78 (1, 24) for the conversion of serine /Xi4D~OH to choline methyl groups and to thymine methyl groups, respectively, have been reported. Conversion of THFA-CH20H to THFA-CHO and back to THFA-CH,OH would result in a ratio of 0.50. Repetition of this process would not be expected to decrease the ratio further, since the hydrogen with a single spatial distri- bution would be removed and added in each oxidation and reduction. Since the THFA-Ci4D0 may be highly diluted by a pool of unlabeled citrovorum factor, the THFA-C14DHOH produced from it may contribute relatively little Cl4 or deuterium to methyl groups, compared with THFA- VD,OH directly formed from serine. Under these conditions the THFA- CH,OH and THFA-CHO could undergo extensive interconversion with little effect on the ratio of Ci4: D dilution of the thymine or choline methyl groups.

The participation of citrovorum factor in purine synthesis and breakdown in pigeon liver has been indicated by recent reports. Buchanan and Schul- man (25) observed a stimulatory effect of leucovorin on the incorpora- tion of radioactive formate into inosinic acid in a pigeon liver enzyme system. Greenberg (26) later observed t,he formation of a substance from leucovorin and ATP that converted 5-amino-4-imidazolecarboxamide to inosinic acid. Flaks and Buchanan (27) have reported the stimulation of the reverse process by the natural citrovorum factor, glycine, and copper. The conversion of both the serine a-carbon and formate to carbons 2 and 8 of purine involves some process in which a labilization of the carbon- bound hydrogen occurs (24).

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56 SERINE BIOSYNTHESIS

The r81e of homocysteine in serine formation is uncertain. Berg tenta- tively postulated the existence of S-formyl- and S-hydroxymethylhomocys- teine intermediates of formate metabolism to explain the specific stimula- tory effect of homocysteine on the incorporation of formate-Cl4 into serine P-carbon in untreated pigeon liver extracts (10). Dcodhar subsequently found that homocysteine stimulates and cysteine inhibits the incorporation of formate-Cl4 into serine p-carbon in untreated chicken liver extracts.7 Homocysteine does not, however, stimulate the interconversion of serine or glycine or the utilization of formaldehyde and formate in Dowex-treated, dialyzed extracts supplemented with THFA. Studies of Doctor and Couch (18) suggest that homocysteine may be involved in the reduction of folic acid to tetrahydrofolic acid. Homocysteine was reported to in- crease the conversion of folic acid to citrovorum factor; glutathione, ascor- bic acid, and cysteine were without effect.

SUMMARY

The incorporation of glycine and formaldehyde-U4 into serine in Dowex l-treated and dialyzed pigeon liver extracts has been reactivated by the sole addition of 5,6,7,8-tetrahydrofolic acid. Formate-Cl4 incorporation has been stimulated by the addition of tetrahydrofolic acid, together with ATP, DPN, Mn++, and glucoseB-phosphate. The introduction of formaldehyde and formate-Cl4 into serine occurred by net synthesis of the amino acid. The implications of these findings have been discussed.

BIBLIOGRAPHY

1. Elwyn, D., Weissbach, A., and Sprinson, D. B., J. Am. Chem. Sot., 73, 5609 (1951).

2. Sprinson, D. B., Elwyn, D., and Weissbach, A., Abstracts, American Chemical Society, 124th meeting, Chicago, 26C, Sept. 9 (1953).

3. Welch, A. D., and Nichol, C. A., Ann. Rev. Biochem., 21, 633 (1952). 4. Deodhar, S., and Sakami, W., Federation Proc., 12, 195 (1953). 5. Blicke, F. F., in Adams, R., Organic reactions, New York, 1, 303 (1942). 6. Broquiat, H. P., Fahrenbach, M. J., Brockman, J. A., Jr., Stokstad, E. L. R.,

and Jukes, T. H., J. Am. Chem. Sot., 73, 3535 (1951). 7. O’Dell, B. L., Vandenbelt, J. M., Bloom, E. S., and Pfiffner, J. .J., /. Am. Chem.

sot., 69, 250 (1947). 8. Melville, D. B., Rachele, J. R., and Keller, E. B., J. Biol. Chem., 169,419 (1947). 9. Siegel, I., and Lafaye, J., Proc. Sot. Exp. Biol. and Med., 74, 620 (1950).

10. Berg, P., J. Biol. Chem., 206, 145 (1953). 11. Chantrenne, H., and Lipmann, F., J. Biol. Chem., 187, 757 (1950). 12. Frisell, W. R., Meech, L. A., and Mackenzie, C. G., J. Biol. Chem., 207,

709 (1954). 13. Robinson, H. W., and Hogden, C. G., J. Biol. Chem., 136, 707 (1940). 14. Blakley, R. L., Nature, 173, 729 (1954). 15. Dietrich, L. S., Monson, W. J., Gwoh, H., and Elvehjem, C. A., J. Biol. Chem.,

194, 549 (1962).

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16. Hill, C. H., and Scott, M. L., J. Biol. Chem., 196, 189, 195 (1952). 17. Wieland, 0. P., Hutchings, B. L., and Williams, J. H., Arch. Biochem. and

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Roy L. Kisliuk and Warwick SakamiSERINE BIOSYNTHESIS

A STUDY OF THE MECHANISM OF

1955, 214:47-57.J. Biol. Chem. 

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