Sept. 5 , 1953
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Dowex-16 and charcoal7 caused a more pronounced difference. With 0.1 ml. of the treated extract (0.86 mg. protein) the complete system yielded 13.5 pM. of carbon dioxide, whereas when either CoA, or ATP or both were omitted, 2.6, 0.9 and 0.2 p1M. of carbon dioxide was produced, respectively. Neither Mg++ nor cocarboxylase affected the rate of the reaction under these conditions. There was no carbon dioxide production when malonate was omitted or the extract was treated a t 100' for 5 minutes . A reaction mixture (prepared as described above but with tris- (hydroxymethy1)-aminomethanebufThe formation of cis-dibromide by either or both fer, flH 7.0, instead of acetate buffer) containing of these paths appears to be unique in the literature. 1.0 ml. of the crude extract and 200 pM. of hy(6) (a) H. Meerwein and K. van Emster, B e r . , 6 3 , 1815 (1920); droxylamine, yielded 7.4 pM. of hydroxamic acid 66, 2500 (1922); (b) P.D. Bartlett and I. Packel, THISJOURNAL, 69, derivatives8 in the presence of ATP and CoA, 820 (1937); 60, 1585 (1938); (c) T. P. Nevell, E. de Salas and C. L. whereas only 0.15 pM. was formed in the absence Wilson, J . Chem. Soc., 1188 (1939). of the added cofactors. These hydrosamic acid deDEPARTMENT OF CHEMISTRY rivatives were tentatively identified by paper UNIVERSITY OF SOUTHERN CALIFORNIAJEROME A . BERSON chromatography (Whatman No. 3 with waterLos ANGELES7 , CALIFORNIA RONALD SWIDLER saturated butanol as solventg) as (1) acethydroxRECEIVEDJULY 1, 1953 amic acid (I?!: 0.51-0.53) and as (2) malonmonohydroxamic acidlo (Rf: 0.36-0.38). Thus the mechanism of malonate decarboxylation PARTICIPATION OF ATP AND COENZYME A I N T H E appears to involve activation of malonate (probENZYMATIC DECARBOXYLATION OF MALONIC ably as malonyl CoA) as a primary step, analoACID gous to the mechanism of succinate decarboxylation Sir : recently proposed for anaerobic microorganisms. 1 1 , 1 2 Malonic acid was previously shown to be an I t has not yet been established whether the deintermediate metabolite of uracil degradation by carboxylation occurs a t the activated carboxyl bacterial enzymes. 2 , 3 More recently decarboxyla- group to form an active one carbon compound and tion of malonic acid was observed with dried bac- free acetate or whether the other carboxyl group is terial cells and crude extracts4 I t has now been decarboxylated to produce active acetate and carfound that the enzymatic decarboxylation of bon dioxide. Since crude extracts were found to malonic acid requires adenosinetriphosphate (AT- form hydroxamic acid derivatives from acetate, P) and coenzyme A (CoA) and an activated form of propionate, and succinate under the conditions demalonate is proposed as an intermediate. scribed above, purification * of the enzymes inPseudomonas jluorescens strain TR-23,6 a strictly volved appears to be necessary to elucidate this aerobic microorganism, was grown for about 20 point. hours a t 26', with constant shaking, in a medium DEPARTMENT O F MICROBIOLOGY containing 1% NHICI, 0.5% disodium malonate, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE 0.15% K ~ H P O I ,0.05% KH2P04, 0.02% MgS04. ST.LOUIS,MISSOURI OSAMU HAYAISHI13 7H20 and 0.1% Difco yeast extract. Cell-free RECEIVED JUNE 16, 1953 extracts were prepared by grinding the washed (6) H. Chantrenne and F. Lipmann, J. Bioi. Chem., 187, 757 (1950). cells with alumina (Alcoa A-301) in the presence of (7) R. K.Crane and F. Lipmann, i b i d . , 301, 235 (1953). reduced glutathione (1.5 mg. of the sodium salt per (8) F. Lipmann and L. C. Tuttle, i b i d . , 169, 21 (1945). g. of wet cells), extracting with 6 parts of 0.02 M (9) Incubation mixtures were treated with Dowex 50 ( H + form) phosphate buffer (pH 7.0), and centrifuging a t and then treated according to E. R. Stadtman and H. A. Barker, J. B i d . Chem., 184, 769 (1950). 25,000 X g for 30 minutes. (10) The author is indebted t o Dr. David Lipkin for suggestions in A reaction mixture (2.0 ml.) containing 0.1 ml. preparing synthetic malonmonohydroxamic acid. of the crude extract (1.43 mg. protein), 100 pM. (11) E. A. Delwiche, E. F. Pharesand S. F. Carson, Federation Proc., KF, 20 pM. reduced glutathione (sodium salt), 12, 194 (1953). 76, 1518 (1953). (12) H. R. Whitley, THISJOURNAL, 10 pM. MgC12, 200 pM. sodium acetate buffer (13) The excellent technical assistance of hlrs. Natalie A. Fraser is (pH 5.8), 100 units CoA, 10 pM. ATP (sodium salt), gratefully acknowledged. 50 y cocarboxylase and 100 pM. sodium malonate was incubated under pure nitrogen a t 30' for 30 minutes. In the complete system 28.8 pM. of A MODEL FOR THE CONFIGURATION O F SULFHYDRYL GROUPS I N PROTEINS carbon dioxide was evolved. When A T P and CoA were omitted, only 1.9 pM. of carbon dioxide was Sir : The differing reactivity of protein S H groups produced. Pretreatment of the extracts with both and, especially, the marked increase in their re(1) This investigation was supported in part by a research grant activity upon denaturation of the protein, has been (097373 from t h e National Institutes of Health, Department of Health, Education and Welfare. the subject of much speculation. We wish to re(2) 0. Hayaishi and A. Romberg, J. B i d . Chem., 197, 717 (1952) port two sets of observations which suggest an (3) F. J. S.Lara, J. B u t . , 64, 279 (1952). explanation for this phenomenon. (4) C. T.Gray, ibid., 63,813 (1952). In the first series of experiments three cysteine(5) 0.Hayaishi and R. Y.Stanier, J. B i d . Chem., 196, 735 (1852).
quiring stereospecific approach of bromine to Cb from the exo-direction, and or (11) the unstable abromoether (XII), ionic rearrangement of which to IIB is in precise steric and electronic analogy to the change camphene hydrochloride + isobornyl chloride.6
