March 20, 1057
COXSIUNICATIONS TO TIIE EDITOR
for the preparation of VIc without further treatment. For analysis a small sample was recrystallized from benzene, The colorless plates melted a t 145-147'.
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Anal. Calcd. for CsHsN03: C, 60.32; H,5.06; N, 7.82. Found: C, 60.25; H , 5.06; N, 7.78. LINCOLN 8, NEBRASKA
COMMUNICATIONS T O T H E EDITOR THE ROLE OF ADENOSINE TRIPHOSPHATE I N T H E ENZYMATIC ACTIVATION OF CARBON DIOXIDE'
Sir: Although many compounds are known to undergo chemical modification or "activation" prior to entrance into biochemical reactions, carbon dioxide has generally been considered to participate simply as such or as bicarbonate ion in a variety of carboxylations in living cells. As recently proposed,2 however, the reaction of carbon dioxide with ATP3 may furnish a reactive intermediate capable of carboxylating HIV CoA (an intermediate in leucine metabolism). Further study of this system has led to the discovery of the bicarbonateand hydroxylamine-dependent cleavage of ATP to AMP and pyrophosphate, catalyzed by extracts of bacteria, yeast and various animal tissues. The reactions shown are proposed to account for this finding and for the mechanism of carbon dioxide activation
+ ATP _r AMP-COz + pyrophosphate AMP-CO2 + SH2OH --+ AMP + (carbonohydroxamic acid?) AMP-CO2 + H I V CoL4If .4MP + H M G CO-4 I1MG CoA z acetoacetate + acetyl Coil CO,
(1)
(2) (3) (4)
The incubation of hydroxylamine and bicarbonate with a heart preparation free of myokinase and pyrophosphatase but containing the carbon dioxide-activating enzyme (H enzyme) results in the degradation of ATP to approximately equimolar amounts of adenylic acid4 and pyrophosphate (Table I). These products would be expected if this enzyme catalyzes the formation of AMP-COS (the mixed anhydride of adenylic and carbonic acids) ackording to Reaction 1, and the intermediate decomposes non-enzymatically according to Reaction 2. Attempts to detect carbonohydroxamic acid have so far proved unsuccessful. However, hydroxylamine has been shown to con( 1 ) Supported b y grants frrmm t h e United S t a t e s Public H e a l t h Service ( G r a n t P;o. A-993C) a n d t h e Michigan Memorial-Phoenix Project of t h e University of Michigan. (2) M. J. Coon, Federation Proc., 14, 762 (1955). (3) Abbreviations: adenosine triphosphate, A T P ; adenosine diphosphate, A D P ; adenylic acid, A M P ; adenosine phosphoryl carbonate, A M P - C 0 z ; 8-hydroxyisovaleryl coenzyme A, H I V CoA; +4-hydroxy-@-methylglutarylcoenzyme A, H M G CoA. (4) Assayed by t h e action of adenylic deaminase according t o 11. M . Kalckar, J . Bioi. Chern., 167, 429 (1947).
vert synthetically prepared AAMP-C028to AMP, and synthetically prepared AMP-C02 ethyl ester to AMP and a heat-labile hydroxamic acid chromatographically indistinguishable from authentic carbonohydroxamic acid ethyl esterg (Rf0.72 in TABLE I F ENZYMES CATALYZIKG THE BICARBONATE-DEPENDENT CLEAVAGE OF A T P The test system contained 5CO pmoles of potassium bicarbonate, 10 pmoles of ATP, 2 pmoles of Versene, 50 pmoles of magnesium chloride, and, where indicated, crystalline pyrophosphatase5 (0 3 mg. of protein), 10-fold purified H enzyme (2.3 mg. of protein) or 30-fold purified F enzyme (1 .O mg. of protein), and 200 pmoles of neutralized hydroxylamine or 300 pmoles of potassium fluoride, in a final volume of 3.0 ml. Incubation, 30 minutes a t 38". In control experiments, 500 umoles of tris-(hydroxymethy1)-aminomethane buffer, pH 8.1, was substituted for bicarbonate.
SEPARATION OF H
AND
orthophosphate formed6
uhfoles pyrophosphate formed7
0.04
0.76
uMoles
Additions
H enzyme
+ + + + +
SH20H H enzyme NHzOH pyrophosphatase H enzyme KF F enzyme NHzOH F enzyme KF
+
1.50 0
0.02 0 0 0 (1.97 pmoles of fluorophosphate")
a Estimated colorimetrically7 using an authentic sample of fluorophosphate as a standard. The absence of pyrophosphate mas demonstrated by paper chromatography.
water-saturated butanol). The H enzyme has been completely separated in this laboratory from fluorokinase (F enzyme), the enzyme in heart extracts which has been shown by Flavin, Castrohlendoza, and Ochoa'" to catalyze the fluorideand bicarbonate-dependent cleavage of ATP to ADP and fluorophosphate.ll These investigators have proposed10 that the F enzyme may activate carbon dioxide for propionyl CoA carboxylation.12 As indicated in Table I , the H enzyme has no signi( 5 ) Kindly furnished by Dr. M . Kunitz. (6) Estimated according t o C. H. Fiske and Y.SubbaRow, J . B i d . C h e m . . 66, 375 (1925). (7) Estimated according t o R . If. F l y n n , M. E. Jones a n d F . Lipmann, J . B i d . Chem., 211, 791 (1951). (8) B. K. Bachhawat J. F. Woessner and AT. J. Coon, Federation Proc., 15, 214 (1956). (9) L. W. Jones, A7n. Chevc. J . , 20, 1 (1898). (10) M. Flavin, H. Castro-hfendoza a n d S. Ochoa, Biochi~i. E L biophys. acta, 20, 591 (1956). (11) This compound was incorrectly identified a s pyrophosphate i n a n earlier report.2 Fluorophosphate a n d pyrophosphate are readily distinguishable from orthophosphate b u t not from each o t h e r b y colorimetric test7 a n d by paper electrophoresis iu citrate buffer, pII 9.0. (12) R f . Flavin, P. J. Ortiz and S. Ochoa, S o l r c r e . 176, 823 (1955).
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COMMUNICATIONS TO THE IZDLTOK
ficant activity in the fluoride assay system, and the F enzyme has none in the hydroxylamine assay system. I n the absence of hydroxylamine, Reaction i can be coupled with Reaction 3 (catalyzed by HIV CoA carboxyla~e'~) to yield HMG CoA. The action of the H M G CoA cleavage enzyme14 converts this product quantitatively to equimolar amounts of acetyl coA4 and acetoacetate (Reaction 4). The data in Table I1 demonstrate that the H
OPTICAL ROTATORY DISPERSION STUDIES. X.' DETERMINATION OF ABSOLUTE CONFIGURATION OF or-HALOCYCLOHEXANONES'
Sir: The determination of the absolute configuration of carbonyl compounds by rotatory dispersion
measurements',~involves a comparison of the rotatory dispersion curve of the unknown substance with those of reference ketones (or aldehydes) which have been sterically related to D-glyceraldehyde. In connection with an extensive study5 T A B L E 11 of the effect of a-substitution upon the rotatory dispersion curves of ketones, there has been made C A R B O N DIOXIDIl PIXATIOS 13Y THE COLPI,V,l7 .ICTIC)S O F !I ENZYME A N D 0-I-IYDROXYISOVALERPL Co.1 CARBOXYLASE an observation which appears to offer a means of establishing the absolute configuration of cycloThe test system contained 590 pnioles of potassium bihexanones without requiriiag a r e f e r e ~ c ecompound carbonate, 5 pmoles of Versenc, 50 ptnoles of magr.esiu:n chloride, 10 pmoles of glutathione, 20 pmoles of ATP, 2 of known abso?ute conjiguration. pmoles of HIV CoA, and, where indicated, H enzyme (2.3 While introduction of an equatorid halogen atoiii mg. of protcin), F enzyme (2.0 mg. of protein), a n d a pig (chlorine, broiiiine but ?Lot fluorine) in the aheart fraction containing HIV CoA carboxylase but free of II and F enzymes ( 5 . 2 ing. of protein), in a final volume position of a cyclohexanone does not seem to affect the sign of the rotatory dispersion curve of the of 3.0 ml. An excess of HMG Co.4 cleavage enzyme was present in each experiment. Iiicubatiori was for 60 minutes halogen-free parent k e t ~ n ethis , ~ is not necessarily a t 38". H I V CoA was omitted in control experiments. the case with axial bromine or chlorine substituents. I n the latter instance, the stereochernistry pMoles acetoEnzymes a d d e d
H enzyme F enzyme H enzyrnc
acetate f o r m e d l
+ Carboxylase + Carboxylase
0.60 0 0.07 0.02
Carboxylase
enzyme is active in this system whereas the F enzyme is without effect. It is concluded, therefore, that the H enzyme and the carbon dioxideactivating enzyme are probably identical, but that the F enzyme plays no role in carbon dioxide activation for HIV co.4 carboxylation. I n accord with these findings, the coupling of Reactions 1, 3 and 4 in 3 heart extract free of pyrophosphatase has been found to furnish approximately equimolar amounts of pyrophosphate and acetoacetate. Since the results presented establish two enzymatic steps in the carboxylation of HIV GoA$, it seemed of particular interest to determine which of these might he lacliirig in biotin deficiency."j Enzyme extracts prepared from the livers of biotindeficient rats possess no H I V CoA carboxylase activity, but have thc same content of H enzyme, F enzyme, and HMG CoA cleavage enzyme as do those preparcd from norinal 1 i ~ e r s . l The ~ H enzyme has recently been purified GSO-fold from pig heart extracts and found to require the presence of Zn++ ions for iiiaxiinal activity. (13) l3. K. 13achharvdt, W. C. Robinson and M. J. Coon, J. Bioi. Chem., 219, 539 (1956). (14) B. K. Bachhawat, W. G. Ilohinsoil aud M. J . Coon, ibid., 216, 727 (1955). (15) Estimated b y a modilicdtion of l h e method of S. S. 'Barkulis a n d A. L. Lehninger. J. Biol. Chewi.. 190, 339 (1951). (10) C. W. E, Plaut a n d H. A. L a r d y , J. Biol. Chcm., 186, 705 (1950).
(17) Unpublished experiments carried o u t b y t h e a u t h o r s in collaboration with Dr. J. F. Woessner a n d Dr. H e n r y A. Lardy.
DEPARTMENT O F BIOLOGICAL CIILMISTRY MEDICAL SCHOOL R. K. BACHIUWAT UNIVERSITYOF MICHIGAN M. J. COON ANN ARBOR,MICHIGAN RECEIVED JANIJARY
81, 1957
of the
o s ij I -c-c-
chronlophore as a whole becomes
dominant and controls the sign o j the Cotton eJect. The gross shape of the dispersion curve can be predicted by examining a model of the appropriate ketone in the following nianner : Place the cyclohexanone ring in such a way that the carbonyl carbon atom is the "head" of the chair and look along the 0-C bond as shown by the arrow in I. As demonstrated in Table I , if
Axial a-haloketone
Cotton effect Halogenfree a-Halohetoue ketone
3a-llroiiioandrostan-?-otlc- 176-01 propionate Positive 7a-Bromocholestane-~13,ja-diol-6-one3-acetate h-egative 7ol-Broniochoiestatie-Xil,.ja-diol-0-one 3,S-diacetate Negative tj~-Bromocholestan-.'{3-~~l-7-one acet3te Negatiic Ba-Rromoergostan-3~-oi-ll-oucacetate I'osi t ive 15ol-Bromoergostan-~~-O~-ll-one acetate I'ositive 12~-Chloro-ll-ketotigogeninacetate Positive 12a,S3-Dibrorno-ll-ketoti#ugeajn acetate Positive I r e t h y 1 1 I p - b r o m t ~ - 3 aacetoxp-12-ket[,cliol~~natePositive Sa-Bromolriedelin Kegativc la-Bromofriedelin Negative
Positive l'osi ti v e Positive Positive Positive h-egative Negative Segative Negative Negative l'usitive
the axial a-chluriiie or broiiiiiie atom lies to the right (ca. 105' angle') of the observer (I, X = Cl or Ur), the single Colton effect ,will be positiue; if (1) Paper I S , C. Djcrassi and W. l i l y n e . Chcirtirfry a n d I % d u s t v y , 988 (195G). (2) Supported b y G r a n t No. CY-PO19 f r o m t h e National Cancer I n s t i t u t e , National Institutes of Health, U. S. Public H e a l t h Service. (3) C. Djerassi, R. Riniker a n d B. Riniker, T I ~ SJouRNAr., 7 8 , 0363 (1950). (-1) C. Djerassi, e l ai., to be published. T h e rotatory dispersion curves of t h e ketones listed i n Table I will be reproduced in t h a t article. ( 5 ) For example, cholestan-3-one, Sa-bromocholestanone a n d 4 a broniocholestanone all exhibit a positive single Cotton effect curve.' ( 0 ) F o r nomenclature in rotatory dispersion work see C. Djerassi a n d W. Blyne, Proc. C h r ? ~Soc,, . 5 5 (1957). (7) It. C. Cookson, J. Chcin. SOL.,181 (1954).
