2384
Vol. '74
COMhIUNICATIONS TO THE EDITOR
salt of a-lipoic acid, prepared in a similar manner, was amorphous. Biosynthetic studies4 provided the initial evidence favoring the straight chain carbon skeleton. A mutant strain of Escherichie coli, which can synthesize a-lipoic acid but which requires a more complex form of this factor for growth, produces appreciably more a-lipoic acid in the presence of acetate or more particularly 2,4,6-octatrienoic acid. This effect was observed in resting cell suspensions and was favored by the presence of cysteine. mCaprylic acid and pyruvate mere inactive in these tests. Further confirmation of this structure was obtained by comparing the infrared spectrum of a-lipoic acid with that of various fatty acids. The infrared spectrum revealed no resolved methyl absorption a t high dispersion in the 3.4 X region. I t has thus been established that the carbon skeleton of a-lipoic acid is the straight chain Cx acid, and a-lipoic acid is therefore the intraniolecular disulfide of a dimercapto-M-octanoic acid, uiisubstituted in the a- and $-positions. The optical activity of crystalline a-lipoic acid was found to he ; U ] ~ I -4-Xi.7' I (1.W; in benzene. 141 I,. J Reed and 13. C D ~ H I I unpul~lisherl ~, rewliI j I O C H E M I C A I . I SSTITUT 1)EPARTMEST OF C H E X I . * S I V E R S I T Y O F TEXAS, CLAYTOSFOUSDATIOAFOR RESEARCH
organic phosphate from ATP, In the presence of fluoride, ATP was found to be converted to an acid-labile phosphate, identified eventually as inorganic pyrophosphate, and to adenosine monophosphate (AXP). Fluoride preserves the pyrophosphate by inhibiting pyrophosphatase, which is a Contaminant of our enzyme, A balance experiment is shown in Table I. Was determined by the hexokinase-hexose~norlophosphate-dehydrogenase-TPN procedure of Kornberg,G ALZP spectrophotometrically according to Kalckar7 using Schmidt's deaminase. Pyrophosphate was determined by manganese precipitation according to Kornberg.6 The pyrophosphate was further identified by the use of a five times recrystallized pyrophosphatase,8kindly supplied to IIS bv Dr. Kunitz. I-ABLE
1
1xicl1 vessel contained: 29 p M ATP, 250 pM acetate, 8W PM NHlOH (pH 6 EJ), 80 pM glutathione, 160 pM potassium fluoride, 640 p M tris-(hydroxymethyl)-amhomethane buffer (PH 7.4), and 32 pM MgCl? in 3.2 ml. total volume. Each vessel contained 0 32 rnl of the yeast enzyme CoA, units
Incubation time, min.
APSI TP,
..\LIP, ,m
Acethydroxamic acid, pT\I
Pyrophosphate, p M
LESTERJ. REED
ACSTIX, TE.Y.IS LILLYR E S E A R C H LABORATURIES QUESTIN I;. SOPER " \'slue determined with pyrophosphatase ELI LILLYASD COMPASY GEORGE 15. F. SCHNAKEYBERG ISDIASAPOLIS, ISDIAY.\ S T A S L E V F. K E R N HAROLD Roaz In view of these results, the ATP-CoA-acetate
reaction is formulated as a two-step reaction
P A K T M E A T O F UAC.l.bRIOLIJCY IVERSITY O F ILLISUIS RBASA, I L L I S O I S R E C E I V E D ? I ~ A R c H 29,
+ CoA eAMP + CoA-pyrophosphate CoA-pyrophosphate + acetate eacetyl-coA -k ATP
1952
pyrophosphate ENZYMATIC PYROPHOSPHORYLATION OF COENZYME A BY ADENOSINE TRIPHOSPHATE'
Sir: I t is well known that acetate may be enzyinatically activated by a reaction chain involving adenosine triphosphate (ATP) and coenzyme A (CoA). Xcetyl-CoA was found to be the product of this reaction,2 the mechanism of which became of parhave shown ticular interest since Lynen, et ad.,3~4 acetyl-coA to be an acetyl mercaptoester. We studied the -4TP-CoA-acetate reaction with an enzyme obtained from yeast extract by protamine and arnrnoniuni sulfate fractionation. The generation of acetyl-Coil was followed by the use of hydroxylamine as chemical acetyl-acceptor, determining acethydroxarnic acid, according to L i p iiiann and Tuttle." .In important lead toward the iirirlerstanding of the mechanism was obtained when it was found that acethydroxamic acid may : m u mulate without an equivalent liberation of iri( 1 ) This work was supported by the National Cancer Institute of the Sational Institutes of Health, Puhlic Health Service; the ritomic Energy Commission; a n d the National Foundation for Infantile Parnly&. 1 3 ) T. C. Chou and F. 1,iptn:inii. .l. B i d . Chein , 196, 89 (1952). t,:j) I' I.ynen and 1 ) . R r i c h e r t , . Cheiii., 63, 17 (19.51) r - i ) 1; T,ynen, E. Keiiliirt :iud 1.. Kueff, .4;r;I., 514, 1 (1951). ~ i 1;.i J , i j i n i : i i i i i and 1 I- L ' i i i i l c . , 1. i(it,! i'.'luiii., 158. .iOT, ( 1 9 4 . 7 ) .
(,
1)
(2)
The exchange of pyrophosphate for acetyl in acetyl-CoX as indicated by reverse reaction (2) was confirmed in the following experiment : T A B L E 11 Each sample contailled, per j ml,: 1.8 $1 acetyl-CoA, 200 p M potassium pyrophosphate or arsenate or phosphate, 200 p M magnesium chloride, 100 p l l potassium fluoride, 1 ml. yeast enzyme. Incubate 30 minutes a t 37". The pH was 7.1. pLI acetyl-CoA"
Initial liicubated with pyrophosphate Substituted arsenate for pyrophosphatc Substituted phosphate for pyrophosphate a 1)etertniried as acethvdroxamic acid.
1.8 U .
