reported from several laboratories. These “oxygenases”2 are generally concerned with the transformation (hydroxylation, ring cleavage, cyclization) of various ring compounds such as aromatic amino acids, phenols and steroids. I n this communication we wish to report a novel type of decarboxylation reaction in which atmospheric oxygen is incorporated into a simple aliphatic compound. L-Lactate was incubated with a crystalline lactic oxidative decarboxylase prepared from JIycobacterium phlei3 in an atmosphere uf 0 2 l s and with H2016 as a medium. Potassium acetate isolated from the incubation mixture was found to have incorporated approximately one atom of atmospheric oxygen (Table I). The OI8 enrichment of the incorporated oxygen atom corresponded to approximately 82y0of that of the atmospheric oxygen used in this experiment, assuming only one atom was inserted into the product. On the other hand, when the reaction was carried out in the medium of H201S and with O2I6 as a gas phase, potassium acetate isolated did not contain appreciable Ols. Carbon dioxide did not contain 0 l s in the first TABLE I ESZIWATIC IXCORPORATIOS ore 0 ’ 8 m r o ACETATE Expcriment I : Crystalline lactic oxidative decarboxylase3 (3!350 units), 2 millimoles of DL-lithium lactate and 2 inillimoles of potassium phosphate buffer ( p H 6.0) were incubated in a total volume of 50 ml. of v a t e r in a special flask designed for this type of experiment.a An 0218-helium gas mixture ( 2 : 3 ) was used as a gas phase.* Experiment 11: T h e same reaction components as in Experiment I were cmployed except t h a t HzOi8 was used as a solvent and 0 2 1 6 helium gas mixture ( 2 : 3 ) was used as a gas phase. The incubation was carried out a t 37” for 30 minutes with vigorous shaking. After the oxygen and carbon dioxide xere removed for mass spectrometric analyses, the incubation mixture was immediately chilled a t 4’ and pH was adjusted to 3.5 with several drops of 2?; H z S O ~ . T h e solution i m s then frozen and water and acetic acid were distilled from the frozen state. The distillate was neutralized with 1iY KOH t o p H 6.5 aud water n-asremoved by lyophilization. The residue dried over P2Oj was pyrolyzed“ and the 0 ’ 6 content determined with a mass spectrometer.d Bxpt.
Type
Atom Yo excess
Cpd. ;inalyzed
case, but was highly enriched in the second experiment. This incorporation of 0ls from HzOI8into carbon dioxide probably was caused by a nonenzymatic exchange reaction as reported by Cohn and Urey?and more recently by Rothberg and Stein-berg in their studies of various microbial decarboxyla3es.j The enzyme which catalyzes “oxidative” decarboxylation of L-lactate has been described froin various microorganisms.6 The over-all reaction involves oxidation and decarboxylation of L-lactate to form stoichiometric quantities of acetate and carbon dioxide, but the mechanism of this unique enzymatic reaction has not yet been completely understood. The available evidence suggests that a t least one atom of atmospheric oxygen was incorporated into acetate when the C1-C2 bond of lactate was cleaved and that the other atom of the oxygen molecule probably was reduced to water CH-CHOH-COOH
+
09’8
--+ CHjC00”H
+ CO? -t I-IrO“
The mechanism of this reaction may be analogous to that of the so-called “mixed function oxygenase”lFJ except that instead of reduced pyridine nucleotides, the substrate itself is providing elcctrons to reduce one atom of oxygen. (4) R I . Cohn and H. C . Urey, T a m J o c ~ i i a ~60, . , 679 (1W8). ( 5 ) S. Rothberg and D. Steinberg, ibid., 1 9 , 3274 (10.571. ( 6 ) (a) S . L. Edson, Biochem. J , 4 1 , 143 (19-17); (b) S. I,. 1311sull and F. B. Cousins, S a t w e , 171, 702 (10Z3): ( c ) Y.Yamamura, XI. Kusunose a n d E. Kusunose, z 6 i d . , 170, 207 (1992); J . Biochein , J Q p Q l Z ,39, 227 (1932); (d) W B . Sutttlvr, F r d i ’ i n r , 1 6 , c t niopliys. A r l i i , 2 0 , 2fi9 (I%;(\); 302 (l!15(!); ( c ) l i , 1;. l k c r i , J I , ,A‘,!otr(, 111, 71, ( 2 ) I , A fIvl,pc!, 1’ 1 O r t i / : ~ 1 1 , 1 S ~ I ~ ~ I i\u i. ,
Sept. 5 , 1957
COMMUNICATIONS TO THE EDITOR
phoretic mobilityS and by enzymatic assay with the pyruvic kinase ~ y s t e m . Both ~ ADP and ,4TP had the expected specific activity, based on that of P3*-labeled Pi used in the incubation. No ADP was formed in the absence of Pi. Inorganic pyrophosphate was not required, and in fact was rapidly hydrolyzed to P i by the liver fraction. AMP was not an intermediate in the formation of ADP from Poly A. Thus, with 50 ug. of enzyme and conditions similar to those of Table I, these quantities of P3?-labeled Pi were esterified (in pmoles) : (1) 0.035 with Poly A equivalent to 0.4 pmole of adenine, in 3 hours; ( 2 ) 0.000 with 0.5 pmole of AMP, in 3 hours; ( 3 ) 0.000 with 0.1 pmole of 9 M P and 0.02 pmole of ADP, in 11 hours.
481 1
sues suggest that nucleoside 5’-triphosph ates are the substrates for polymerization. The exact function of the phosphorolysis reaction in RNA metabolism is under investigation. NATIONAL ISSTITUTE OF ARTHRITIS R . J . HILMOE AXD METABOLIC DISEASES L. A . HEPPEL XATIOSALISSTITUTES OF HEALTH UNITEDSTATES PUBLIC HEALTHSERVICE BETHESDA, MARYLAND RECEIVEDJUXE 19, 1957
EXPONENTIAL KINETIC DEPENDENCIES I N INHIBITED AUTOXIDATIONS’
Sir:
The conventional treatment of the induction ti, in inhibited autoxidations has been based upon the steady-state assumption with reA mixture of 1.0 mg. of liver fraction, 19.3 pmoles of - ~assumpPO1--- buffer (PH 7.2, 95,000 c.p.m. of P32 per pmole), spect t o chain carrier c ~ n c e n t r a t i o n , ~an tion which accounts well for the observed direct Poly A* equivalent to 6.8 pmoles adenine and 9 pmoles of MgClz, all in 0.9 nil., was incubated at 37’ for 12 hours. proportionality between inhibitor concentration Nucleotides were adsorbed by norite in the presence of and induction p e r i ~ d . ~Furthermore, ,~ the nonperchloric acid and the washed suspension mas eluted with linear relationships between ti and inhibitmsr cona n ethanol-h7H40H-water mixture. Aliquots of the eluate centration, obtained for stabilized samples of were assayed eiizymatically for ADP, counted for P32,and chromatographed on paper for quantitative separation of cracked gasolines, have been fitted to an empirical AMP, A D P arid ATP. equation? which is isomorphous with that deduced Amount Specific activity through a steady-state treatment of a simple chainReaction product rmoles c.p.m. per omole branching reaction ~ c h e m e . ~ XMP~ 0.80 110 There is, however, a significant body of minter-1DP 0 , s5c 80,000 preted observations of accelerating increases in ti ATPb 0.45 16~,000 with increasing inhibitor concentrationsg and o€ Total esterified Pi 1.75“ decelerating decreases in t i with increasing cona Synthesized from ADP, usirig polynucleotide phoscentrations of pro-oxidant catalyst^.^^^^ These obphorylase from A . vinclandii ( l a ) , A T P and part of the A M P were formed by adenylate kinase. T h e remaining servations have not been explained in terms of From A M P was formed from Poly A by a nuclease. steady-state kinetics. absorption a t 260 nip after chromatographic separation; We wish to report the obtainment of well-defined 0.85 pmole of A D P was also found by enzymatic assay.4 From sum of A D P and 2 X .4TP. Total esterified Pi exponential dependencies of ti upon both a.ntioxidant and catalyst concentrations. The data shown determined by direct count of the washed norite suspension was 1.9 pmoles. in Table I were obtained with purified tetralin a t The liver enzyme had a pH optimum a t about 70’ using a manometric apparatus.“ These data pH 7, required Mg++ and was inhibited completely conform to the relationship t i = t i 0 exp (kX) by 0.06 M fluoride. K m was 3 X 10-3M for P i in (1) the phosphorolysis reaction. The enzyme also where X is the concentration variable The catalyzed the exchange of 0.5 ,umole of P32-labeled least-squares values for k and the standard deviaP i with ADP per hour per mg. of p r ~ t e i n . ~For tions are: set A, X = dibutylcresol concn , k = for Mg++, 2 X this reaction K m was 1.3 X 0.87 kg./millimole, s.d. & 0.01; set B, X = 10-4JI for ADP and 3 X 10-3X for Pi. An ex- cobaltous naphthenate concn., k = - 0.257 change of Pi with ATP was also noted, so far un- kg./micromole, s.d. =I= 0.003; set C, X = cobaltous explained. Experiments with other nucleoside naphthenate concn., k = - 0.100 kg./micromole, diphosphates must await removal of an interfering (1) Presented in part before the Division of Organic Chemistry phosphatase (inactive with ADP), a t t h e 131st meeting of the American Chemical Society, Miami, Although phosphorolysis of adenylic polynu- Florida, April, 1957. This work was generously supported by a from T h e Research Corporation cleotide to give ADP has been demonstrated here grant ( 2 ) P. George, E. K. IZideal and A. Robertson, PYOC.R 3 y . S O L , for the first time in animal tissues, net synthesis of 8185, 288 (1940). polynucleotide could not be detected (even with (3) P. George and A. Kobettson, ; D i d , 309. (4) G. S. Hammond, C . E . Buozer, C . E. Hamilton a u d J. K . Sen, C’*-ADP) because of contaminating nucleases. Previous results on the incorporation of CI4- T a I S J O U R N A L , 77, 31’38 (1935). ( 5 ) G. W. Kennerly a n d UT.L . Patterson, Jr., “Kinetic Sludies of labeled ATP6l7and UMPs into RNA of animal tis- Petroleum Antioxidants,” 128th meeting of the American C!hemical TABLE I period, PHOSPHOROLYSIS OF ADENYLIC POLPSUCLEOTIDE
+
(3) K . Markham a n d J , D. Smith, Biochem J . , 62, 552 (1952). (4) A. Kornberg a n d 1%’. E. Pricer, Jr., J . B i d . Chem., 193, 481 (1951). ( 5 ) T h e same activity per mg. of protein was obtained by measurn g t h e rate of phosphorolysis of Poly A a n d this value was 5 % of t h a t obtained with a purified E . ccli fraction ( I b ) . ( 6 ) P. C . Zamecnik, IC.I,. Stephensun, J. I?. Scott and M. B. Hoagland, F e d . Pvoc., 16, 27.5 (1957). 17) I
