7652
COBIMUNICATIONS T O THE
TABLE I SUBSTRATES FOR THIOESTERASE 15-fold purified chicken liver thioesterase. -0.5 p J I thioester in 0.067 M Tris-HC1 buffer p H 7.0. Specific activity = -AE.uo per minute per mg. protein. Values it1 parentheses refer t o synthetic S-acetyl ester. Specific activity
8-S-AcAc-BITO 82 ( )-8-S-AcAc,F-ethyl-MTO 69 ( f)-S-AcAc-DTO 56 ((i,0)" ( )-6-S-AcAc-MTO 17 ( rt )-G-S-Ac Ac-Decanoate 7.5 S-AcAc-BAL 310 (30) (+)-6-S-Ac-DTO was hydrolyzed a t same rate as t h e synthetic ester.
* *
EDITOR
Vol. 79
(or Pn). Tlic prccise role of D'TO :mrl other thiols in activating hcAicsynthesis is being investigated further. DEPARTMENT OF PAARMACOLOCY h'f.EDICINE GEOR(:EI DRLJXMO\I> WESTERNRESERVEUNIVERSITY Jossri~R STERU CLEVELAND 6, OHIO I I ~ U R U A 27, R V 19-17 SCHOOL O F
A NEW PATHWAY FOR PROPIONATE OXIDATION
Sir: Oxidation of propionate in animal tissues occurs by a carboxylation pathway through methyl malonate to succinate. Stadtman2 has observed the formation of P-alanyl-CoA from acryl-Co.l extracts of Clostridium propionicurn. llahler and Huennekens suggest a11 a-oxidative pathway. I n this communication evidence is presented i l l support of a @-oxidative pathway in peanut initochondria. The oxidation of sodium propionate-l-Ol4 to CI4O2 by mitochondria isolated from cotyledons of germinated peanuts4& is dependent upon ATP,,5 CoA, D P N , GSH and QKG; T P N and l l t i + . ' stimulate the oxidation.
thioester and one mole of thiol are formed. This stoichiometry is consistent with reaction 3 and follows from the fact t h a t AcAc-SR does not assay as thioester by the hydroxylamine method5 while S-Ac-DTO assays as both thiol and thioester. The Ac-S-Pn and Ac-S-DTO formed were further identified by paper chromatography. I t is not yet determined whether 6- or 8-S-Ac-DTO is formed. This mixed thiolysis reaction represents a novel enzymatic synthesis of S-Ac-DTO. It differs from the synthesis of (+)-6-S-Ac-DTO from Ac-S-CoA and (- )-DTO catalyzed by DTO transacetylase7~* TAULE I in that it involves transacetylation with a 4-carbon fragment and utilizes both (-)-DTO and (+)- COFACTOR REQUIREMESTS F O R OXIDATION O F P R O P I O S A T E I-cl4TO c1402 DT0.13 The enzyme(s) catalyzing reaction 3 difThe coriiplete reaction mixture coiitaiiied 0.1 piiiole propiofcrs from the t h i o l a ~ e ' of ~ ~the ' ~ fatty acid cycle (re(5500 c.p.In.) ; 0.5 r n l . mitochoiitlria (approxiaction 1) in that (a) AcAc-S-Pn is more reactive 1iate-1-C'~ 20 tng. protein) in 0.2 -11 Tris- 0.5 31 sucrose, p € l than AcAc-S-CoA, (b) it is less sensitive to iodo- mately 7 . 2 , containiiig about 5 X l o r 3 yo B;\L; 10 pmoles phos:wetainide, and (c) it is not readily reversible, if a t phate buffer, pH 7.1; 50 pinoles KCI; 1 puiole ATP; all. Interestingly, other thiol compounds which 0.3 pmole Co.4; 0.2 pmrilc U P S ; 0.1 ptnole T P N ; 5 pnioles GSH; 1 ptnole a K G : 0.2 nil. 20% KOH in the activate AcAc svnthesis (e.g., R.\L, GSH, cysteine) ceiiter well; 0.3 inl. 10 .lf H2SOl i i i the sideurni, final volcxn substitute for DTO in reaction 3 , yielding the utnc 1.7 tnl. Tiiiie OF iiicuhation, 2 h r . , tcrril~erature25". corresporiding S-Ac ester. 4 0 1 ( c 1) 111.) '< l O ( ~ / s 1 l > ~ t r (act .ep . i x ) , l~\l'I,'l,PX
21
~'ln~mll?nl,
''2
OXld.ll1011
>
21 1;' 31
I 1
)
I
-'l'lj.Y .(>SII
Since pools of pyruvale, lactate, succinate a ~ c l methyl nialonate added during propionate-l-CI4 oxidation do not acquire any label, these c m l pounds do not appear to participate as intcrmediates. Furthermore, no propionate-dependent fixation of C1.'02 can be demonstrated. T o examine the course of oxidation, propioiiatc(+)-S-AcAc-DTO is reduced by D P X I in the presence of crystalline heart @-hydroxybutyryl-S- l-CI4, -2-C'* and -3-CI46 were incubated with L: CoA dehydrogenase.'S Liver fractions do not cata- complete reaction mixture fur different periods of lyze a thiolysis of (*)-S-AcAc-DTO by DTO, CoA- time and ether-extractable reaction products sepSH, Pn-SH or GSH. Nor do they convert (+)-6- aratcd by papcr chromatography. In each case a new radioactive spot (Rf 0.23 in ethanol alnrnonia; S-Ac-DTO' or (&)-S-S-Xc-DTO to AcAc. Since thioesterases exist in liver for S-AcAc-DTO propionate Rf 0.42) appeared, which decreased arid AcAc-SG, the latter are possible intermediates (1) hI. Flavin, P. J. Ortiz and S. Ochoa, .Vature, 176, 623 (19k55). in AcAc synthesis. However, experiments so far ( 2 ) E. R. S t a d t m a n , Federalioiz Proc., 16, 3lio (1956). (3) H. R . hZahler and F. hi. IInennekens, B i o c h i m et HioPhy3. Acta, have failed t o demonstrate their enzymatic formation, e.g., by AcAc transfer from AcAc-S-COB 11,(4)575I n(1953). a recent personal communication. LJr. SI. J. Coon describes a Acetyl acceptor
(zk)-DTO
A AcAc-S-PI>"
-1.50 4 Sulfhydryl' 4-1.43 4-2.99 A Hydroxamic acid Measured optically at 310 mp.
(+)-DTO
(-)-DTO
-1.50 -1.50 4-1.44 4-1.71 4-2.98 +2.88 Xitroprusside assay.
(13) We are indehted to l k , 1;.I'olkers of hIerck-Sharp and Ilohme I:ecearch 1,aboratories for (+) and ( - ) lipoic acids. (14) J. K. Stern and S. Ochoa, in "Biochemical Problems o f Lipids," I j u t t e r a o r t h s Publications, 1,ondon. 1956, p. 162. (15) 1.'. Lynen, K . Decker, 0. Wieland a n d D. Reinwein, ref. 11, 1'. 1.41'. ( 11;) J . R . Stern, unpublished experiments.
- -
propionate BIiP $-alanine pathway i n nnimal tissues. I n peanut mitochondria n o &alanine accumulates. (4a) P. R. Stutnpf, Plant Physiolory. 3 0 , R5 (1933). ( 5 ) Abbreviations: ATP,adenosine triphosphxte; C n 4 , C O ~ I I Z V I ~ P .\: D P N , diphosphopyridine ntlcleotide; GSII, glutathione; \In++, manganese; aRG,a-ketoglutarate; OIIP, P-hydroxypropionnte (6) Kindly donated b y Dr. Harland G . Wood.
COMMUNICATIONS TO THE EDITOR
May 20, 1957
with time. This spot, identified as PHP, was characterized by (a) two dimensional co-chromatography with authentic PHP, (b) dichromate oxidation' of PHP derived from propionate-2-C14, to a dicarboxylic acid which co-chromatographed with malonic acid. The dicarboxylic acid was degraded by pyrolysis* to radioactive acetate and COSand (c) esterification of the acid with diazomethane and conversion to a hydroxamic acid (with hydroxylamine) which co-chromatographed with authentic PHP hydroxamate. Both propionate-l-C14 and PHP-1-C14 rapidly release the ~arboxy1-C'~ as CI4O2. No radioactive Krebs cycle acids are produced. The cofactor required for the oxidation of PHP-1-C14 to C1402is ATP. Under the same conditions, propionate-2-C14 and /3HP-2-C149slowly release C14 as C1402,only after the appearance of radioactive citric, succinic, malic and fumaric acids. With propionate-3-C14 a rate of CI4O2release between t h a t of propionatel-C14 and of -2-C14 is observed with the formation of labeled Krebs cycle acids. Succinate derived from propionate-l-C14 is not labeled. Succinate derived from propionate-2-C14 is labeled exclusively in the methylene groups and succinate derived from propionate-3-C14 is labeled exclusively in the carboxyl groups. The conversion of propionate-C14 to PHP-C14 is dependent on oxygen, ATP and CoA. Other cofactors were not tested. The pathway may be formulated as1O CHa.CH2.COOH
ATP, CoA
CH,=CH.CO,
C H ~ , C H Z , C O . C O+ A HzO
COA +CHzOHCHz.CO.CoA
I CH,OHCH,.COOH/
'
7 -
HOOC.CH8
1 Krebs CycleI Acid
f
+-
IATP
,coz,
(7) By the method used for lactate oxidation in S. Aronoff, "Techniques of Radiobiochemistry," The Iowa State College Press, Ames, Iowa, 1956, p. 141. (8) S. Aronoff, ref. 7, p. 140. (9) Radioactive BHP, presumably labeled as shown, was isolated by paper chromatography of the reaction product of propionate-1-C" or -2-C'4 oxidation. (10) Compounds in blocks were isolated and characterized. (11) This work was supported in part by a grant from the National Science Foundation.
DEPARTMENT OF AGRICULTURAL BIOCHEMISTRY COLLEGE OF AGRICULTURE J. GIOVANELLI OF CALIFORNIA P. K. STUMPF'' UNIVERSITY BERKELEY 4, CALIFORNIA RECEIVED MARCH27, 1957
2653
THE STEREOSPECIFIC RADICAL ADDITION OF HYDROGEN BROMIDE TO cis- AND t~~ns-2-BROMO-2BUTENE'
Sir: I n previous work it was found that a high degree of stereospecificity is observed in the radical addition of hydrogen bromide to c y c l o h e ~ e n eand ~~~ cyclopentene4 derivatives. From this and other considerations it appeared possible that under favorable conditions stereospecificity might be observed with this addendum in acyclic systems. We wish to report that the radical addition of hydrogen bromide to the isomeric 2-bromo-2-butenes in liquid hydrogen bromide a t -80" is indeed stereospecific and results in almost complete trans addition. Other radical additions which have been investigated have been found to be non-stereospecific6and apparently this is the first report of a stereospecific radical addition in an acyclic system. The additions were promoted by irradiation with a quartz-jacketed Hanovia type SC-2537 lamp which fit into the reaction vessel so that the reaction mixture occupied the annular space between the walls of the lamp and reaction vessel. During the reaction the vessel was immersed in a Dry Ice-acetone bath. Mixtures of approximately 20 ml. of anhydrous liquid hydrogen bromide and 5 g. of cis- or trans-2-bromo-2-butene (shown to be homogeneous by gas chromatographic analysiss and physical properties) were irradiated after which the hydrogen bromide was allowed to evaporate and the composition of the residual reaction mixture determined by gas chromatographic6 and infrared analysis. I n a typical experiment pure cis-2-bromo-2butene in liquid hydrogen bromide was irradiated for 7.5 minutes. The composition of the product (gas chromatographic analysis6) was found to be < 0.5% 2-bromo-2-butene (largely isomerized to the trans isomer), 3y0 2,2-dibromobutane (presumably formed by ionic addition), 92y0 meso2,3-dibromobutane, 5y0 dZ-2,3-dibromobutane and < 0.5% 1,2-dibrornobutane. Irradiation of trans-2-bromo-2-butene for 15 minutes under similar conditions gave a mixture consisting of 5y0 of unreacted trans-2-bromo-2butene, 83y0 dl-2,3-dibromobutane, 8.5% meso-2,3dibromobutane and 3.5y0 1,2-dibromobutane. The analytical method was calibrated and confirmed using pure authentic samples of all of the components and synthetic mixtures of these. Infrared spectroscopic examination of the reaction products also showed t h a t the cis-bromobutene is converted primarily to meso-dibromide and the (1) This work was supported by the United States Air Force through the Air Force Office of Scientific Research of the Air Research and Development Command under contract No. AF 18(600)1037. (2) H. L. Goering, P. I. Abell and B. F . Aycock, Jr., THISJOURNAL, 74,3588 (1952). (3) H.L. Goering and L. L. Sims, i b i d . , 77, 3465 (1955). (4) K.L. Howe, unpublished work. (5) P. S. Skell and R. C. Woodworth, THISJOURNAL,77, 4638 (1955); P. S. Skell, R. C. Woodworth and J. H. McNamara, ibid., 79, 1253 (1957). (6) Excellent separation was obtained with a 10 ft. column packed with the commercial detergent, Tide. An operating temperature of 70' and flow rate of 40 ml. helium per minute were used to check for intercontamination of the isomeric 2-bromo-2-butenes. An operating temperature 120' was used for analysis of the reaction mixtures.
