COMMUNICATIONS TO THE EDITOR
May 20, 1957
SPECIFIC
ACTIVITYO F
TABLE I DEGRADATION
THE
c l4-SQUALENE
PRODUCTS O F
Degradation product
Specific activity, c.p.m. per mg. C
Acetone Carbonyl carbon CHI3 Succinic acid -COOH -CH2Levulinic acid CHI3 (carbon 5) -COOH (carbons 1 and 4 ) -CH2- (carbons 2 and 3 )
306 10 360 70 30 80 150 17 28 310
&Ha
I
CHD
CoA involves the intermediate formation of AcAcS-CoA by thiolase, which catalyzes reaction 1 . followed by deacylation of AcAc-S-CoA t o A C A C . ~ - ~ However, a direct deacylation of synthetic AcAc-SCoA is not catalyzed by these enzyme fractions. 2Ac-S-Cor\
XcXc-S-Co;\
+ HS-CoA
(1)
We find that AcAc synthesis by partly purified ox or chicken liver enzyme preparations from ilc-
and i t is probable from the data that only one of the methyl carbons of the acetone fragment contained C14 (Fig. 1). C "H3C=CHCH2C*H2C=CHCH2C*HzC=CHCHs-
[
265 1
1
S-CoA (generated by phosphotransacetylase from acetyl phosphate and CoA-SH3) requires the addition of a mono- or dithiol compound. Among active thiols, ( j=)-DT06 has the greatest activity, half maximum activation of Acdc synthesis occurring with 1 X M (*)-DTO compared to 3.5 X X BAL and 2 X M GSH. These liver fractions also catalyze the reactions ( 2 ) , (3) (f)-S-.kcAc-DTO
+ H20 +ACAC+ ( f ) - D T O + +
AcAc-S-Pn (or CoA) ( & ) - D T O J_ hc-S-Pn (or Coli) (&)-S-Xc-DTO
(2)
(3)
CHI distribution of isotopic carbon in biosynthetic squalene.
Mono- ( j= ) -S-AcAc-DTO was synthesized by reFig. 1.-Principal acting one equivalent of diketene with (j=)-DTO. Presumably it is the 8-ester, since acetic anhydride The distribution of isotope in the squalene sug- and DTO have been shown to give 8-S-Ac-DTO.' gests that the mevalonic acid was not decarboxyl- While DTO does not give a positive nitroprusside ated to yield a five carbon intermediate prior to assay for sulfhydryl,8~9we find the monothiol, monocondensation. If a five carbon intermediate is thioester form does. Thus, monoacylation of DTO first produced, the compound would have to react results in the appearance of one sulfhydryl equivaasymmetrically to give the observed isotope dis- lent (by nitroprusside assay), as well as one thioestribution. This possibility has not been excluded ter equivalent (measured with hydroxylamine or by the data presented here. Whether mevalonic optically a t 240 mp). The synthesis and breakacid is or is not the biological precursor of squalene down of mono-S-acyl-DTO compounds can thereand cholesterol, there appears to be little break- fore be measured by nitroprusside assay. The thioesterase catalyzing reaction 2 is assayed down and re-condensation prior t o polymerization. I t is likely that decarboxylation occurs during or optically by following the decrease in light abafter polymerization. sorption a t X 310 mp or X 240 mp, since ( f ) - S Complete details will be presented in a subse- AkAc-DTOhas an absorption spectrum characterquent publication. istic of acetoacetyl thioesters.l@,ll I t is present in Acknowledgment.-This work was supported in liver but apparently not in other tissues. The cnpart by grants from the National Heart Institute zyme has been purified 40-fold from chicken liver of the National Institutes of Health, the American and shown to be different from AcAc-SG thioesterHeart Sssociation, and the American Cancer So- ase.12 It hydrolyzes S-AcAc and S-A4c esters of ciety. DTO, M T O and BAL (Table I). As measured optically a t X 310 m p , liver and heart DEPARTMENT O F BIOCHEMISTRY SCHOOL OF MEDICINE FRANK DITURI enzyme fractions catalyze a disappearance of AcXcUSIVERSITYOF PENNSYLVASIA SAMUEL GURIX S-Pn and AcXc-S-CoA, provided DTO (or certain PHILADELPHIA, PENSSYLVANIA mono- or dithiols) is added. IVith DTO, the deDEPARTXESTO F BIOCHEMISTRY is BAYLORUNIVERSITY JOSEPH L. RABINOIVITZ crease in E 3 1 0 due to disappearance of XcA%c-SK HOUSTON, TEXAS accompanied by a simultaneous increase in Ezra, RECEIVED MARCH16, 1957 signifying an increase in total thioester concentration. Balance studies (Table 11) show that for each mole of Achc-S-Pn disappearing, two moles of E . R. S t a d t m a n , XI. DoudoroE and I?. Lipmann, J . Bioi. C h e m . , SOME ENZYMATIC REACTIONS O F 6,8-DITHIOLOC- 1 9 1(3), 377 (1951).
TANOIC (DIHYDROLIPOIC) ACID AND ITS ACETOACETIC THIOESTER'
Sir: Evidence has been presented that AcAc2 synthesis in soluble liver fractions from a source of Ac-S(1) Supported by g r a n t s f r o m t h e U. S. Public Health Service
F u n d of t h e Research Corporation. ( 2 ) .4hiire\ i:rtir,n>. li,X-dithioloctanoic (dihydrolipoic) acid, DTO; mr~nvthi(,liict:iii(~i~ :ici 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. l l a h l e r 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 t h a t i t 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 t h a t (a) AcAc-S-Pn is more reactive 1iate-1-C'~ mately 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- 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 GSH; 1 ptnole a K G : 0.2 nil. 20% KOH in the activate AcAc svnthesis (e.g., R.\L, GSH, cysteine) pnioles 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 a ( ct e. p . 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-DTOto 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.
