MECHANISM        OF FIXATION OF CARBON                DIOXIDE      IN
                       THE KREBS CYCLE
   Oxidation of pyruvate       by the Krebs cycle involves its union
with oxalacetate.      Liver in contrast to certain other tissue oxi-
dizes pyruvate in the absence of added Cd-dicarboxylic          acid. It

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was proposed I, 2 that the Cd-dicarboxylic       acid is synthesized in
liver by the reaction of Wood and Werkman3               Cl302 + CHD.
CO +COOH         = C1300H. CHZ. CO. COOH.            The isolation1 of
radioactive a-ketoglutarate,     formed from pyruvate in the presence
of radioactive sodium bicarbonate, proved that there is fixation
of carbon by liver.
   We have determined the location ,of the fixed carbon in CY-
ketoglutarate     by use of CY302. The experimental procedure for
the dissimilation    of pyruvate was that employed by Evans and
Slotinl except that sodium bicarbonate containing 9 per cent Cl3
was used. 30 ml. of medium were used in each of six 125 ml.
Warburg flasks.      The ar-ketoglutarate   was isolated from the de-
proteinated solution by binding it with bisulfite, saturating          the
solution with magnesium sulfate, and removing the citric, malic,
succinic, and fumaric acids by continuous ether extraction for 3
days.     The residue of extraction was freed from sulfite by boiling,
extracted with ether for 24 hours, and the ac-ketoglutarate           was
separated from the pyruvate by precipitation          as the silver salt.
The free acid was obtained by acid ether extraction.          The titrat-
able acidity was equivalent to 0.32 InM and the 2,4-dinitrophenyl-
hydrazone (m.p. 223’, mixed m.p. 223”) 0.28 mM of cu-ketoglutarate.
The acid was oxidized with potassium permanganate to CO2
and succinic acid, CY300H.C0.CHzCHz.COOH                    = PO2 +
COOH .CH,.CH,.COOH.               The Cl3 content of the CO2 and suc-
cinate was determined with the mass spectrometer. Succinate
      1 Evans, E. A., Jr., and Slotin, L., J. Biol. Chem., 136, 301 (1940).
      2 Krebs, H. A., and Eggleston, L. V., Biochem. J., 34, 1383 (1940).
     3 Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O., J.
Biol.     Chem., 139, 365, 377 (1941).
484                                 Letters to the Editors

contained the natural complement of Cl3 (1.10 per cent), whereas
the COz contained a high concentration      of Cl3 (2.20 per cent).
These results prove that all the fixed carbon is in one carboxyl
group of the cr-ketoglutarate.  A possible mechanism of formation
of this acid is shown in the accompanying scheme.
           COOH                                                                    co2
           CO                                      COOH                            +
           (3%                                     co                              COOH

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                                -Hz0                                                                  +&O
                              --+                  CH2             --!%            6Hz              --+
           OH                                      &COOH                           &COOH

           &COOH                                   CH                              CH

           CH                                      C’aOOH                          C’aOOH


                                    COOH                                     COOH
                                    6Hz            .                         CH2
                                H&COOH                   --2%           H&H            +   COz
                              HOCH                                      06
                                    &OOH                                     POOH

   The position of the fixed Cl3 in the a-ketoglutarate     is in agree-
ment with its derivation      from pyruvate    and oxalacetate,      the
initial fixation of CO2 being in the oxalacetate.         The results
clearly indicate that citrate is not an intermediate,   for, if cu-keto-
glutarate was derived from a symmetrical         molecule, the fixed
carbon would be equally distributed    in the two carboxyl groups.
Bacteriology   Section                                                                       H.G.      WOOD
  Agricultural   Experiment                Station                                           C.H.      WERKMAN
  Iowa State College
Departments    of Physiological                   Chemistry       and     Physics            ALLAN   HEMINGWAY
   University  of Minnesota                                                                  A. 0. NIER

Received       for   publication,         April        18, 1941

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