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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 I n 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 i t 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 a t 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 A T P 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)
