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4873
an obligatory intermediate in the process (Figs. 1 and 2). The formula of the precursor pyrrole (Fig. l), suggested on theoretical grounds, is the same as that prop~sed~.~J' for porphobilinogen. 11, which has not been previously described, was synthesized by three different procedures: (1) by exhaustive benzoylation of imidazole propionic acidlofollowed by hydrolysis, (2) by nitrosation of P-ketoadipic acid followed by reduction and (3) by a phthalimide synthesis on 6-chlorolevulinicacid. (8) S. Yanari, J . E. Snoke and K. Bloch, J. B i d . Chem., 201, 561 Other metabolic pathways for I1 seem probable: (1953). for instance, a route by which the a-carbon atom DEPARTMENT OF BIOCHEMISTRY of glycine may be utilized for the ureido carbon JOHN E. SNOKE UNIVERSITY OF CHICAGO atoms of the purines," for the P-carbon atom of CHICAGO, ILLINOIS serine,12Jaetc., since I1 may be considered to be a RECEIVED AUGUST27, 1953 derivative of the a-carbon atom of glycine. Testing the implications of Fig. 2 we have found that 6-AMINOLEWLINIC ACID, ITS ROLE IN THE BIO- the 6-carbon atom of I1 is incorporated into the ureido groups of purines in much higher concentraSYNTHESIS OF PORPHYRINS AND PURINES tion than is the a-carbon atom of glycine in comSirs: parable experiments. This accords with the key We wish to report our finding that b-aminolevulinic acid (11) can replace the two substrates, "ac- steps in the postulated cycle (Fig. 2). tive" s u c ~ i n a t e ~and * ~ *glycines*s * for porphyrin synthesis. It would appear, therefore, that I1 is the source of all the atoms of protoporphyrin as outlined in Fig. 1.
synthesis by ADP.* It may therefore be proposed that in the synthesis of GSH from glutamylcysteine and glycine, phosphorylation of the enzyme by ATP is the initial step. Acknowledgment.-The author wishes to express his appreciation for the facilities provided by Dr. Konrad Bloch and for his interest in the above work, which was supported by a grant in aid from the US.Public Health Service.
COOH
I
coon
iH2 iH2iH2
I
CYCLE
COOH
I
I
CH2 +
I
CH2
io
PROTOPORPHYRIN
pu r ine s e t
'rr
b
- ' --
HOOC-CH2-CH2-i-!-COOH
/""
'\Y
k?N
a
CYCLE
NH.,
"2
6-Aminolevulinic acid (11) (11)
+
Precursor pyrrole
Fig. 1.-The formation of the precursor pyrrole for porphyrins from two moles of a-aminolevulinic acid.
The radioactivity of hemin obtained from either CI4glycine plus unlabeled succinate or CX4succinate plus unlabeled glycine, in duck blood, is reduced by 80 to 90% by the addition of an equimolar amount of unlabeled 11. Also, I1 labeled with "6 or with C14 in the 6-carbon atom formed heme containing the isotope in many times the concentration of that formed from labeled glycine; hemin, synthesized from 0.05 mM. of C14-labeled I1 and from 0.05 mM. of C1*-labeled glycine of equal activity had an activity of 22,000 c.p.m. and 500 c.p.m., respectively. I1 is the decarboxylated product of CYamino-P-ketoadipic acid (I), which can be formed from a condensation of "active" succinate and glycine. The above finding is evidence that I is (1) This work was supported by grants from the National Instituter of Health, United States Public Health Service, from the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council. and from the Rockefeller Foundation. (2) D. Shemin and J. Wittenberg, J . B i d . Chem., 192,315 (1951). (3) D. Shemin and S . Kumin, {bid., 198, 827 (1952). (4) D . Shemin, Abstracts, Amer. Chem. Soc. Meeting, Atlantic City, N. J.. 1952, p. 35c. (6) D. Shemin and D. Rittenberg, J . Biol. Chem., 166, 621, 0.27 (1946). (6) J. Wittenberg and D. Shrmie, {bid.. 181, 108 (1950).
io,-
0 keto- q l u t o t o l d e h y d e
HOOC-CH2-CH2-$-COOH
0
d-keto-glutaric acid
J
HOOC-CH2-CH2-~-CH2Nn2 $-ornino- Ievulinic 0 acid(lT)
I
4 PORPHYRIN
Fig. 2.4uccinate-glycine cycle: a metabolic pathway for the oxidation of glycine.
Further, this succinate-glycine cycle geared with the Kreb's citric acid cycle may account for the serine-glycine r e a c t i ~ n , may ~ ~ Jexplain ~ ~ ~ ~the metabolic reactions of the "one-carbon atom" compounds and provide via the oxidation of the proposed ketoglutaraldehyde to ketoglutaric acid, a pathway by which succinate can be converted to ketoglutarate. Consistent with the possible reversibility of these reactions outlined in Fig. 2 is our earlier finding that isotopic formate is utilized, (7) R. G. Westall, Nature, 170, 614 (1953). ( 8 ) G. € Cookson I. and C. Rimington, ibid., 171,875 (1953). (9) 0. Kennard, ibid., 171, 876 (1953). (10) We wish to thank Dr. H. Tabor for a generous sample of urocanic acid. (11) I. L. Karlsson and H. A. Barker, J . B i d . Chem., 177, 597 (1949). (12) T. Winnick, I. Motins-Claeason and D. M. Greenberg, ibid. 171, 127 (1948). (la) W. W a d , ibid., 176, 519 (1949). (14) D.B h M P l i D , J . B l d . C k m , , 1IP, 297 (1916).
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Vol. 75
albeit poorly, for the methene bridge atoms of protoporphyrin.
The hydroxamic acid formed behaved chromatographically like acethydroxamic, not excluding definitely small amounts of other hydroxamic acids. DEPARTMENT O F BIOCHEMISTRY COLLEGE OF PHYSICIANS AND SURGEONS DAVIDSHEMIN In order to identify the other reaction products, COLUMBIA UNIVERSITY CHARLOTTE S. RUSSELL stoichiometric amounts of CoA were incubated with NEW YORK32, N. Y. citrate and ATP. The characteristic thioester RECEIVED JULY 30, 1953 absorption a t 232 pM3 appeared immediately and, as shown in Table I, equivalent amounts of acetyl COB, keto acid and inorganic phosphate were AN ENZYMATIC REACTION BETWEEN CITRATE, formed. No pyrophosphate was detected as a ADENOSINE TRIPHOSPHATE AND COENZYME A1 product of this reaction which differentiates it furSky: ther from the ATP-CoA-acetate r e a ~ t i o n . For ~ During studies on synthetic functions of pigeon further identification of the acyl CoA, the ATPliver preparations, i t was observed that acetone CoA-citrate enzyme was combined with sulfonapowder extracts and the supernatant fraction of mide-acetokinase5; on incubation of this system pigeon liver homogenate catalyze a reaction beTABLE I tween ATP, CoA, citrate and hydroxylamine which The system contained: 40 pM. citrate, 20 pM. of MgClz, leads to the accumulation of large amounts of hy- 20 pM. glutathione, 10 pM. ATP, 2.6 pM. CoA, twice fracdroxamic acid. Acetone powder extracts of other tionated enzyme and water in $total volume of 2 ml., pH 7.4. livers showed slight activity with ATP, CoA and Incubated 40 minutes a t 37 . Keto acid was measured according to the method of Friedeman and Haugen.B For citrate, while yeast extracts appeared to be inac- estimation of acetyl CoA, after incubation, an equal voltive. The enzyme system was partially purified ume of 2 M NHpOH was added to an aliquot and hydroxamic by ammonium sulfate fractionation and in this man- acid determined as usuaLP Acetyl CoA, Keto acid, ner can be separated from the ATP-CoA-acetate UM. Pi, p M . ILM. System system. shown in Fig. 1, between 18 and 34% 3.0 2.9 2.7 Complete ammonium sulfate saturation, most of the activity 0 0 Xo citrate 0 in the hTP-CoA-citrate reaction precipitates, while 0 0 S o CoA 0.17 the ATP-Co-I-acetate system remains in solution. In a similar manner, fractions were obtained which with citrate, ATP and CoA, acetyl sulfonamide and contained the ATP-CoA-citrate system, but did keto acid were formed in equivalent amounts. The not show a citrate formation on incubation with keto acid was identified as oxalacetic acid by measCOXand oxalacetate. This observation appears to uring the decarboxylation with aniline hydrochlodifferentiate our system from Ochoa's condensing ride at 15". Under these conditions, the product enzyme. The purified system did not react ap- of our enzymatic reaction is decarboxylated like preciably with succinate, malate, aconitate or iso- oxalacetate within 5-10 minutes, while acetoacetate citrate, instead of citrate. The reaction is depend- reacts only rather slowly. It is concluded from these observations that we ent on the presence of magnesium ions. are dealing here with an interaction between ATP, CoA and citrate, which yields acetyl CoA, oxalacetate, ADP and inorganic phosphate: ATP + Citrate HS.CoA I_ p 2.0 ATP - GOA- CITRATE acetylCoA + Oxalacetate ADP f Pi (1) 0 5 YS TEM I The reversibility of the reaction is indicated by incorporation of inorganic phosphate into ATP when ATP, citrate and CoA were incubated with Pi3*. Exchange was found only with the complete LL 1.0 0 system but not by incubation of ATP and Pi3' alone. Preliminary experiments indicate a net synthesis of S YSTEM ATP by reversal of reaction (1) if the glucose-hexog 25 kinase system is added as phosphate acceptor. The reaction described here appears to represent I O 0 10 PO 30 40 50 60 70 a new variant of citrate degradation and synthesis. PERCENT. AMMONIUM SULFATE SATURATION, Various attempts to show a primary formation of Fig. 1.-The assay system contained: 1 p M . K citrate, citryl-CoA which is a plausible intermediary, have 10 pM. MgC12, 10 pM. glutathione, 5 pM. ATP, 32 units so far not given promising results.
'i
+
h
+
I\
CoA, 200 pM. NH20H, enzyme, PH 7.4 in a total volume of 1 ml. Controls were run without citrate. Hydroxamic acid was determined according t o Lipmann and Tutt1e.l The ATP-CoA-acetate reaction was measured by substituting acetate for citrate. (1) This investigation wua aupported by 8 research grant from the National Institutes of Health, Public Health Service. The following abbreviations are UBRd: adenaine ttiphoaphate, ATP: adenosine diphosphate, A D P , Idatganlc phosphate, Pi; and coenzyme A, CoA Or HSCoA. (*& F,Li@maaatrd h 59, TVbtlr?t 2,&?#ai,Chm.,i@(lk 81 (104(1),
BIOCHEMICAL RESEARCH LABORATORY PAUL A. SRERE MASSACHUSETTS GENERAL HOSPITAL FRITZ LIPMANN AND THE DEPARTMENT OF BIOLOGICAL CHEMISTRY HARVARD MEDICAL SCHOOL, BOSTON, MASSACHUSETTS RECEIVED AUGUST3, 1953 (8) E. R . Stadtman, Abst., Am. Chem. Sac., Atl8ntic City, Sept. 14, 32C (1952). (4) F. Lipmann, M. E. Jones, S. Black and R. M . Flynn, TRrs JOWRNAL, 74, 2384 (1952). (5) T.C. Chou and F. Lipmann, J . Bid. Chcm., 188, 89 (1962). (6) T. B, Ffkdrtirrn wad 0,6 ,HrrugQn,I , B i d . Ckrm,, a47, 41& (1~4a)
