ACYL ADENYLATES: AN ENZYMATIC MECHANISM
OF ACETATE ACTIVATION*

By PAUL BERGt
Wita THb TECHNICAL ASSISTANCE OF GEORGIA NEWTON

(From the Department of Microbiology, Washington University School of
Medicine, St. Louis, Misaourt)

(Received for publication, March 9, 1956)

Several different pathways are now known for the activation of acetate.
One of these, found thus far only in certain microorganisms (1, 2), is initi-
ated by the phosphorylation of acetate with ATP! by acetokinase (3, 4),
followed by the transfer of the acetyl group to CoA by the action of phos-
photransacetylase (5-7).

(1) ATP + acetate = acetyl phosphate -+ ADP
(2) Acetyl phosphate + CoA = acetyl CoA + phosphate

In animal tissues (8-11), yeast (12, 13), plants (14), and Rhodospirillum
rubrum (15), another pathway of acetate activation has been demon-
strated. This involves a reaction of ATP, acetate, and CoA, resulting in
a split of ATP with the formation of acetyl CoA, A5P, and PP, and has
been termed the aceto-CoA-kinase reaction (12, 16).

(3) ATP + acetate + CoA = acetyl CoA -+- A5P + PP

Analogous reactions with higher fatty acids have also been reported and
characterized (17-19).

In a recent study of the mechanism of Reaction 3, Jones, Lipmann, Hilz,
and Lynen (20) reported that a partially purified enzyme preparation from
yeast catalyzed an exchange of PP and ATP in the absence of acetate and
CoA. They also found that acetate-C' exchanged with the acety] group of
acetyl CoA in the absence of A5P and PP. To account for these observa-
tions, the following mechanism was proposed (20).

* This work was supported by grants from the United States Public Health Service
and the National Science Foundation.

¢ Scholar in Cancer Research of the American Cancer Society.

1The following abbreviations have been used: adenosine triphosphate, ATP;
adenosine-5’-phosphate, ASP; uridine triphosphate, UTP; inosine triphosphate,
ITP; guanosine triphosphate, GTP; cytidine triphosphate, CTP; coenzyme A, CoA;
inorganic pyrophosphate, PP; di- and triphosphopyridine nucleotide, DPN and TPN;
trichloroacetic acid, TCA; tris(hydroxymethyl)aminomethane, Tris; flavin adenine
dinucleotide, FAD; uridine diphosphoglucose, UDPG; nicotinamide mononucleo-
tide, NMN;; flavin mononucleotide, FMN; uridine-5’-phosphate, USP.

991
992 MECHANISM OF ACETATE ACTIVATION

(4) ATP + enzyme = enzyme — A5P + PP
(5) Enzyme — A&P + CoA = enzyme — CoA + A5P
(6) Enzyme ~ CoA + acetate = acetyl CoA + enzyme

Since the existence of the postulated enzyme-bound substrates was
inferred solely from isotope exchange experiments, we investigated this hy-
pothesis once more to obtain further information on the nature of the inter-
mediates and the mechanism of their formation. With a more highly puri-
fied enzyme than that previously employed, it has been found that the
exchange of PP and ATP does not occur unless acetate is added.
This, together with the absence of any exchange of A5P and ATP in the
presence of CoA alone, as predicted by Reactions 4 and 5, indicates that the
proposed mechanism is untenable. In the present paper we wish to present
evidence in support of another mechanism of acetyl CoA synthesis. This
involves a primary reaction of ATP and acetate to form a hitherto unde-
scribed compound, adenyl acetate (shown in the accompanying diagram),
and its subsequent reaction with CoA to form acetyl CoA (Reactions 7

and 8).
O
Po O- 1
|
CH—CH—CH—CH—CH.—0O—Pt-—-O0—-C—-CH;

|

a iN O-
LP

NH,

 

Adenyl] acetate has been synthesized and demonstrated to react enzymat-
ically in the manner shown below.

(7) ATP + acetate = adenyl acetate + PP

(8) Adenyl acetate + CoA = acetyl CoA + A5P

A preliminary communication of this work has been reported elsewhere (21).
The synthesis, purification, and characterization of adeny] acetate are de-
scribed in the following paper (22). The apparently analogous formation
of an amino acid acyl adenylate in the reaction of t-methionine with ATP

by an enzyme from yeast has been briefly mentioned earlier (21) and will be
reported in detail in an accompanying paper (23).

Materials and Methods

PP? was prepared by heating NasHPQ, at 225° for 18 hours (24), and
purified by anion exchange chromatography. ATP, labeled with P® in the
P. BERG 993

terminal pyrophosphate group, was prepared either by exchange of PP
with unlabeled ATP by using purified aceto-CoA-kinase (Reaction 7) or
from A5P and P® by rat liver mitochondria in the presence of a-ketoglu-
tarate. Adenine-C'*-labeled ATP was prepared by first coupling ad-
enine-C with 5-phosphoribosyl pyrophosphate (25) and converting the
A5P-C to ATP-C with adenylic kinase (26), phosphopyruvate, and pyru-
vate phosphokinase (27). The ATP was purified both by adsorption and
elution from Norit and by anion exchange chromatography (28). Acetyl
CoA was prepared from acetyl phosphate and CoA by purified phospho-
transacetylase (5), and partially purified by anion exchange chromatog-
raphy by using a 2 per cent cross-linked Dowex 1 Cl- column and eluting
with 0.1 Nn HCl-0.1 m KCl. According to the optical density at 260 mu
and acetyl CoA assay (11, 29), it was 50 per cent pure. Sodium
acetate-1-C' was obtained from Tracerlab, Inc., and sodium fluoroacetate
was kindly supplied by Dr. R. O. Brady. CoA (75 per cent pure) and CTP
were obtained from the Pabst Brewing Company, and DPN, TPN, ITP,
UTP, and GTP from the Sigma Chemical Company. Reduced CoA was
prepared with potassium borohydride by the method of Jones eé al. (13).

Hexokinase was prepared by a modification of the method of Brown? and
contained 5 units per mg. (1 unit forms 1 ymole of glucose-6-phosphate per
minute at pH 8.0 and 25°). Glucose-6-phosphate dehydrogenase was ob-
tained from the Sigma Chemical Company and contained 0.8 unit (30) per
mg. Crystalline condensing enzyme (31), recovered from the mother liquor
of the first crystallization, was generously given by Dr. 8. Ochoa. This
preparation, when used in the coupled assay for acetyl CoA (11, 29), con-
tained malic dehydrogenase in adequate excess. The amine-acetylating
enzyme of pigeon liver (acetone fraction) was prepared according to Tabor
et al. (82). Crystalline inorganie pyrophosphatase (33) was generously
supplied by Dr. G. Perlmann and Dr. M. Kunitz. Phosphotransacetylase
was prepared by the method of Stadtman (5), and A5P deaminase by Pro-
cedure A of Kalckar (34), adenylic kinase according to Colowick and Kal-
ckar (26), and pyruvate phosphokinase by the method of Beisenherz
et al. (35).

A solution of 2 m hydroxylamine was prepared daily by neutralizing a
4 m solution of hydroxylamine hydrochloride with KOH. More concen-
trated solutions of hydroxylamine were made by adding solid barium hy-
droxide to a solution of hydroxylamine sulfate until the pH was about 7.2.
The barium sulfate was removed by centrifugation and the supernatant
fluid was concentrated under reduced pressure. The volume was adjusted
so that the concentration of hydroxylamine was 5 M, based on the initial

* We are grateful to Dr. D. H. Brown for giving us this method before its publica-
tion.
994 MECHANISM OF ACETATE ACTIVATION

amount of hydroxylamine sulfate. The solution was kept at 4° for periods
of no longer than 2 weeks.

Acetyl CoA was measured by DPN reduction by the citrate-condensing
enzyme system containing malic dehydrogenase (11, 29), and ATP was
determined with hexokinase, glucose, glucose-6-phosphate dehydrogenase,
and TPN (36). Adenyl acetate was determined either by conversion to
acetyl CoA, measured as mentioned above, or by conversion of P?P® to
ATP (see Table VI). Adeny]l acetate concentration was also determined
as acethydroxamic acid after treatment with hydroxylamine. The sample
was incubated for 5 minutes at 37° in 1 ml. of 0.2 m hydroxylamine, and
0.5 ml. of acidified ferric chloride (13) was added and the optical density at
540 mp measured. Under these conditions 1 pmole of acethydroxamic acid
has an optical density of 0.630. This was in good agreement with the
values determined enzymatically (22). Protein was measured by the
phenol method of Lowry et al. (37).

Assay of Aceto-CoA-kinase

Formation of Acetyl CoA—The assay procedure was essentially that de-
scribed by Jones ef al. (13). The reaction mixture contained, in 1.0 ml.,
0.1 M potassium phosphate, pH 7.5, 0.005 m MgCl, 0.01 m ATP, 0.05 m
potassium fluoride, 0.01 m glutathione, 0.15 mg. of CoA (equivalent to 0.1
umole), 0.01 m potassium acetate, 0.2 m neutralized hydroxylamine, and the
enzyme. The mixture was incubated for 20 minutes at 37°, then 2 ml. of
an acidified ferric chloride solution (13) were added. After centrifugation
to remove precipitated protein, the optical density at 540 my was deter-
mined with a Beckman model DU spectrophotometer. In all cases, a con-
trol tube in which CoA was omitted was incubated with each amount of
enzyme. The formation of 1 umole of acethydroxamic acid resulted in an
increase in optical density of 0.325, and the unit of activity was defined as
the amount of enzyme catalyzing the formation of 1 wmole of acethydrox-
amic acid in 20 minutes under these conditions. As previously shown, the
formation of acethydroxamic acid was proportional to enzyme concentra-
tion in the range of 0 to 0.5 unit (13).

Exchange of P?P®? with ATP—The standard assay mixture contained, in
1.0 ml., 0.1 mM potassium phosphate, pH 7.5, 0.005 mM MgCle, 0.002 m ATP,
0.001 m acetate, 0.05 m fluoride, 0.002 m P?P® containing between 10‘ and
105 ¢.p.m. per umole, and the enzyme. When the purified enzyme was
used, the fluoride was omitted, since there was no demonstrable inorganic
pyrophosphatase activity. The mixture was incubated for 20 minutes at
37° and the reaction stopped by the addition of 0.5 ml. of 7 per cent per-
chloric acid, followed by 0.2 ml. of a suspension of acid-washed Norit (15 per
cent, dry weight). After 3 minutes, the Norit was centrifuged, washed
P, BERG 995

three times with 3 ml. portions of water, and then suspended in 3 ml. of 50
per cent ethanol containing 0.3 M ammonium hydroxide. An aliquot of the
suspension was plated and the P® content determined with a Geiger-Miiller
counter. The activity is expressed as the micromoles of P#P® incorpo-
rated into ATP and is calculated by dividing the total P® activity in ATP
by the specific activity of the initia] PP. 1 unit of enzyme activity is
defined as the incorporation of 1 umole of P®P® into ATP in 20 minutes
under these conditions. Comparison of the values obtained by this pro-
cedure with those found by chromatographic separation (28) of ATP showed
that they were in good agreement. Thus in one experiment the total counts
per minute in ATP determined by the Norit procedure were 6000 (com-
plete), 520 (no acetate), and 500 (no ATP), as compared with the respective
values of 6700, 360, and 600, obtained by chromatographic analysis.

The assay was proportional to enzyme concentration in the range of 0 to
0.4 unit. Thus with 0, 0.59, 1.5, 3.0, and 7.3 y of enzyme protein, 0, 76,
80, 80, 69 units per mg. of enzyme were calculated to be present.

Results

Purification of Aceto-CoA-kinase—All the operations were carried out at
4°. Pressed bakers’ yeast (200 gm.), obtained from Fleischmann’s yeast,
Standard Brands Incorporated, was mixed to a paste with 200 ml. of cold
0.1 m dipotassium phosphate and treated in a 10 ke. sonic oscillator (Ray-
theon) for 40 minutes. The mixture was centrifuged in a Servall centrifuge
at 10,000 X g for 30 minutes, and the turbid supernatant fluid was filtered
through glass wool to remove particles of fat. This crude extract (Table I)
was stable for at least 2 days when kept at —15°.

To 180 ml. of the crude extract were added, with stirring, 27 ml. of a 2 per
cent solution of protamine sulfate, generously supplied by Eli Lilly and
Company. After 5 minutes, the solution was centrifuged for 5 minutes at
10,000 X g and the precipitate discarded. To the clear supernatant fluid
were added 55 ml. of the protamine solution, and after 5 minutes the mix-
ture was centrifuged as above. The supernatant fluid was discarded and
the gummy precipitate washed twice with a total volume of 100 ml. of cold
water by homogenizing with a motor-driven pestle and then centrifuging.
The washing was repeated as described above, 100 ml. of 0.01 m potassium
phosphate, pH 7.0, being used. The washings were discarded and the
precipitate was dissolved in 125 ml. of 0.25 m potassium phosphate, pH 7.5
(protamine eluate).

To the protamine eluate (125 ml.) were added 97 ml. of a solution of am-
monium sulfate, saturated at 4°. After 10 minutes, the mixture was cen-
trifuged for 5 minutes at 10,000 X g and the precipitate was dissolved in
65 ml. of cold water (Fraction AS-1). To this solution was added alumina
996 MECHANISM OF ACETATE ACTIVATION

Cy gel (38) (1.1 mg., dry weight, of gel per mg. of protein) and after 5 min-
utes the gel was centrifuged and washed with a total of 100 ml. of water,
then twice with a total of 100 ml. of 0.05 m potassium phosphate, pH 6.9.
The enzyme was then eluted with two 40 ml. portions of 0.1 m potassium
phosphate, pH 7.5 (Cy gel eluate).

To the Cy gel eluate (80 ml.) were added 21 gm. of ammonium sulfate
and, after 5 minutes, the precipitate was separated by centrifugation and
dissolved in 14 ml. of 0.5 M potassium phosphate, pH 7.6 (Fraction AS-2).

In several trials the purification achieved in the AS-2 fraction in relation
to the crude extract ranged from 35- to 60-fold, and the yield was between
25 and 35 per cent. Fraction AS-2 has been repeatedly frozen and thawed

 

 

 

TABLE I
Purification of Aceto-CoA -kinase
Fraction ner its Total units coneenteation eens,
me. per mi. ~~ fens
Crude extract....... 49 8820 21.4 2.3
Protamine eluate.... AT 5875 2.0 24
AB-1. 0... ccc ee. 59 3835 1.9 31
Cy gel eluate........ 35 2800 0.42 | 83
AB-2. cic eae 157 2190 1.6 98

 

 

* Assay, formation of acetyl CoA, measured by formation of acethydroxamic acid
as described in ‘‘Methods.”’

several times a week for periods of up to 4 months without appreciable loss
of activity.

P®P® Exchange with ATP—It had been reported previously (20) that
aceto-CoA-kinase catalyzed an exchange of P#P® and ATP in the absence
of acetate and CoA. Since such a reaction could have occurred by other
previously reported reactions (36, 39, 40) involving endogenous substrates,
and therefore could be unrelated to the formation of acetyl CoA, the ex-
change reaction was again examined. It was found that, in addition to
ATP, P#P*, and Mgt, acetate was required for the exchange reaction with
the most purified fraction of aceto-CoA-kinase (Table II). There was no
appreciable exchange when either ATP, Mg*, acetate, or the enzyme was
omitted. The requirement for acetate became increasingly apparent with
purification; in one experiment the ratio of activities in the presence and
absence of acetate was 4 in the crude extract, 60 in Fraction AS-1, and 143
in Fraction AS-2.

The rate of exchange as a function of acetate concentration is shown in
Table III. Since the réle of acetate is catalytic, small amounts of acetate
P. BERG 997

effect an appreciable amount of exchange of P?P*? with ATP, the extent of
which is dependent on the time of incubation. It therefore seems likely
that the previously reported exchange reaction (20) was due, at least in
part, to the presence of acetate and perhaps to other compounds unrelated
to acetyl CoA formation. The inhibition of acetate at a concentration of
3 X 10-3 Mm and higher has not been observed with acetyl CoA formation as

TABLE II
Requirements for P33P" Exchange with ATP

 

 

Components P2P2? incorporated into ATP
pmole
Complete. ........ cece cece eee eee ees 0.55
Minus ATP............. 00. ccc eee 0.01
2 Mgt cae 0.01
f€ acetate... ee cece eee 0.02
© ENZYME. 2... ee eee 0.00

 

Usual assay procedure for PP-ATP exchange with 0.002 m acetate and 7.5 + of
enzyme (Fraction AS-2); specific activity 92.

 

 

Tase III
Effect of Acetate Concentration on P#®P**-ATP Exchange
Concentration P"P# incorporated into ATP

X 104m pmole

0.0 0.02

0.4 0.15

2.0 0.39

10.0 0.57

30.0 0.43
40.0 0.37

 

 

The conditions are the same as for the experiment described in Table IT.

a measure of the aceto-CoA-kinase reaction. The specificity of the reaction
for acetate (see below) and the effectiveness of small amounts in the ex-
change reaction suggest the use of this reaction for measuring acetate in the
range of 10-* to 10°-° Mm.

Time-Course and Extent of Exchange—The exchange of P®P# and ATP
approaches the calculated value for complete equilibration of the pyrophos-
phoryl group of ATP and the P#P* (Fig. 1). ATP prepared in this way
contains essentially equal amounts of P* in the two terminal phosphate
groups (41). Since the enzyme preparation did not contain any adenylic
kinase activity as measured with ATP and A5P in the presence of pyruvate
998 MECHANISM OF ACETATE ACTIVATION

phosphokinase and lactic dehydrogenase (42), the reaction appears to rep-
resent an exchange of the two pyrophosphate moieties.

Specificity of Nucleotide and Fatty Acid Components—ATP was the only
nucleoside triphosphate which was active in the exchange reaction. UTP,
CTP, ITP, and GTP did not replace ATP. Formate, butyrate, caproate,
and octanoate at concentrations of 1 to 2 X 10-3 m did not replace acetate.
Propionate, the only other fatty acid activated by the aceto-CoA-kinase
(16), also catalyzed the exchange reaction. At 5  10-' Mo, the rate was

 

OL. Theoretical Maximum

Fae ae ee a ee

SOL

umole P3?PX%in ATP
S § 8 8 B 8
T T T T T

oe

Ss

 

 

tL ! f { ] 1
0 30 50 70 % HO BO 150
Minutes
Fre, 1. Time-course and extent of PP-ATP exchange. Reaction mixture: 1.0
ml. containing 0.10 M potassium phosphate buffer, pH 7.5; 0.005 m MgCl; 0.0016 m
ATP; 0.001 mM potassium acetate; 0.002 m P#P* containing 5 &X 10! c.p.m.; 4 of en-
zyme, specific activity 96. Temperature 37°.

 

about 30 per cent that found with 1 x 10-?M acetate. With fluoroacetate
at levels of 1 X 107? m and 5 X 10-* M, there was about 2 and 5 per cent
the exchange found with 1 X 10-? m acetate. Whether this is a property
of fluoroacetate or due to contamination by acetate is uncertain. There
was no significant inhibition of the exchange when equimolar amounts of
acetate and fluoroacetate were present. Brady (43) has reported that
neither yeast nor liver aceto-CoA-kinase converts fluoroacetate to fluoro-
acetyl CoA but that kidney preparations do.

Formation of Acethydroxamic Acid from ATP and Acetate—The observa-
tion that acetate was required for the PP-ATP exchange suggested that the
initial step in acetyl CoA synthesis was a reversible enzymatic interaction
of ATP and acetate with the formation of adeny] acetate and PP (Reac-
P. BERG 999

tion 7). ‘This hypothesis was tested with hydroxylamine to trap the acety!
group of adenyl acetate as acethydroxamic acid. With large amounts of
enzyme, ATP, and acetate, and high concentrations of hydroxylamine
(2.5 m), there was a net formation of acethydroxamic acid (Table IV), which
increased with time and was dependent on the presence of ATP, acetate,
and the enzyme. When a smaller amount of hydroxylamine was used,
e.g. 1M, there was only 16 per cent as much acethydroxamate formed. The
rate of acethydroxamic acid formation was also dependent on the amount
of enzyme; thus, with 33, 82, 164, and 330 y of enzyme, there was a forma-

 

 

 

 

 

 

TaBLe IV
Formation of Acethydrozamic Acid fram ATP and Acetate in Absence of CoA
Time
Components |
30 min. | 60 min. 120 min.
umole A umole | 4 umoles ray

Complete......... 0.52 0.30 | 0.89 0.69 | 1.60 1.38
Minus acetate....; 0.22; ; 0.20 | 0.22

“ enzyme....| 0.04 : ' 0.04 | 0.04 |

“ATPL. 0.04 0.04 | 0.04 |

 

All the values are expressed in micromoles of acethydroxamic acid and the change
(A) was calculated from the difference between the values obtained in the presence
and the absence of acetate. Reaction mixture: 1.0 ml. contained 0.1 m potassium
phosphate, pH 7.5; 0.005 ma MgCl,; 0.01 m ATP; 0.01 m acetate; 2.5 m hydroxylamine;
and 330 y of enzyme (specific activity 96); temperature 37°. The reaction stopped by
the addition of 0.2 ml. of acid (2 N TCA, 6.6 n HCl), followed by the addition of 0.5
ml. of ferric chloride reagent (12).

tion of 0.04, 0.12, 0.27, and 0.58 umole of acethydroxamic acid in 60 min-
utes.

Acethydroxamic acid formation under these conditions was accompanied
by the liberation of an equivalent amount of A5P and PP (Table V). In
another experiment, with 412 y of enzyme, A5P formation was measured
at the end of the incubation with A5P deaminase. In the presence of ace-
tate, 1.1 umoles of acethydroxamic acid and 1.3 umoles of A5P were formed,
and, in the absence of acetate, 0.1 umole of acethydroxamic acid and 0.1
umole of A5P were found.

The above results are in agreement with the postulated formation of
adenyl acetate as an intermediate, an interpretation, however, dependent
on the assumption that small amounts of CoA were not present. In order
to determine whether CoA was present, a sensitive assay for CoA was de-
veloped which involved the use of high concentrations of hydroxylamine
(2.5 M) in the usual acetyl CoA assay system. By developing the color in a
1000 MECHANISM OF ACETATE ACTIVATION

volume of only 1.5 ml., as little as 0.0005 umole of CoA could be detected.
Heat-inactivated samples of the enzyme (330 y) tested in place of CoA in
the above assay gave no detectable amounts of acethydroxamic acid in
either the presence or the absence of glutathione. Asa control, CoA added
to the enzyme solution before or after heating was quantitatively recovered.
Moreover, when a sample of heated enzyme (330 y) was added to the reac-
tion mixture (Table IV), there was no increase in the amount of acethy-
droxamic acid over that found in the presence of 330 y of unheated enzyme
alone, whereas the addition of 0.0005 umole of CoA doubled the formation
of acethydroxamic acid. These experiments indicate that the acethy-

TaBLE V

Stoichiometry of Products Formed in ATP-Acetate
Reaction in Presence of Hydrozylamine

 

 

Components ancethy: A Pp* A ASP{ | 4
I
umole pumoles umoles
Complete,........ 0.74 0.71 1.11 0.76 1.12 ; 0.73
Minus acetate.... 0.09 0.06 0.45 0.10 0.46 0.0
“««  enzyme.... 0.03 0.35 0.39 |

 

* Calculated from the original specific activity of ATP.

t Calculated from the optical density at 260 mu, with an extinction coefficient of
16 X 103 cm.-! wv"! (44). Reaction mixture: 1.0 ml. contained 0.1 m Tris, pH 7.4,
0.005 m MgCl:; 0.0077 m P%*-labeled ATP containing 3.31 X 104 ¢.p.m. per zmole;
0.01 mM acetate; 2.6 M hydroxylamine; 330 y of enzyme (specific activity 125). Incu-
bated 120 minutes at 37°. Heated aliquots of the incubation mixture were chroma-
tographed on Dowex 1 Cl- columns to separate A5P and PP (24). The amount of
acethydroxamic acid formed was determined colorimetrically in a parallel experi-
ment, as described in Table IV.

droxamic acid formed in the absence of added CoA is not due to endogen-
ous traces of CoA. This conclusion is further supported by the observa-
tions reported in the section pertaining to A5P exchange with ATP in
which both acetate and added CoA were required for the exchange. These
experiments, therefore, are consistent with the formulation of adenyl
acetate as an intermediate in acetyl CoA synthesis.

Utilization of Adenyl Acetate for ATP Synthesis—From the experiments
discussed thus far, it was considered likely that the intermediate formed
from ATP and acetate was the phosphoacetyl derivative of A5P. This
compound was synthesized first from the silver salt of A5P and acety] chlo-
ride by a modification of the method of Lipmann and Tuttle (45), but better
yields and purer material were obtained by direct acetylation of A5P with
acetic anhydride by the method of Avison (46). These are presented in
detail in Paper II (22).
P. BERG 1001

Synthetic adeny! acetate was converted to ATP in the presence of PP,
Mgt*, and aceto-CoA-kinase (Table VI). This was shown with P®P® and

 

 

TasLe VI
Formation of ATP from Adenyl Acetate and PP
Components | Time PP incorporated into ATP

min. umole
Complete........0..0....... | 20 0.23
cece cece 45 0.28
Minus enzyme.............. 20 | 0.01
Complete*.................. 20 0.00

 

* The adeny! acetate was previously incubated in 0.1 m KOH for 5 minutes at room
temperature and then neutralized to pH 7.0. Reaction mixture: The volume of 1.0
ml. contained 0.1 M potassium phosphate, pH 7.5; 0.005 m MgCl.; 0.002 m P#2P3?,
0.28 umole of adenyl acetate (measured by hydroxamic acid assay); and 7.2 y of
enzyme (specific activity 72).

Be.)

 

32eL
28). +aATP

LOL

Ly moles

081

 

 

i ! ! t
Q 30. 40 SOQ 60 7
Minutes

Fiae. 2. Correspondence of ATP formation and adenyl acetate disappearance.
Reaction mixture: 1.0 ml, containing 0.1 mM potassium phosphate buffer, pH 7.5;
0.005 m MgCl.; 0.00032 m adenyl acetate; 0.002 m P??P3?: and 7.2 7 of enzyme (specific
activity 72). Temperature 37°. ATP determined by Norit assay and adenyl acetate
by hydroxamic acid assay.

1 L

0 2

 

by measuring P® in ATP after adsorption and elution from Norit. Accord-
ing to these data, ATP was formed in an amount equivalent to the amount
of adenyl acetate added. Treatment of adenyl acetate with dilute alkali,
which rapidly hydrolyzed adenyl acetate to A5P and acetate (22), destroyed
this activity. The formation of ATP (Fig. 2) occurs concomitantly with a
1002 MECHANISM OF ACETATE ACTIVATION

disappearance of adenyl acetate as measured by hydroxylamine-labile
acetyl groups. An over-all balance of the formation of ATP from adeny]
acetate and PP has already been presented (21).

The enzymatic conversion of adenyl acetate to ATP has also been verified
spectrophotometrically by being coupled to the phosphorylation of glucose
with hexokinase. Incubation of adenyl acetate, PP, Mgt+, and aceto-
CoA-kinase in the presence of an excess of hexokinase, glucose, glucose-6-
phosphate dehydrogenase, and TPN resulted in a reduction of TPN which
was proportional to the amount of adenyl acetate added (Table VII). The

TABLE VII

Formation of ATP from Adenyl Acetate and PP Measured Spectrophotometrically
with Hexokinase and Glucose-6-phosphate Dehydrogenase

 

 

Adeny] acetate added* j TPNH formed
umole pmolet
0.000 0.000
0.024 0.025, 0.025, 0.027
0.0387 0.039, 0.040, 0.038
0.054 \ 0.056
0.084 . 0.085

1

* The adenyl acetate concentration was determined by acethydroxamic acid for-
mation and by conversion to acetyl CoA as measured with the malic dehydrogenase -
citrate-condensing enzymes (10, 29).

¢ An extinction coefficient of 6.22 * 103 em.~'u~! (47) was used. Reaction mix-
ture contained, in 1.0 ml., 0.05 m Tris, pH 7.4; 0.001 m MgCl.; 0.025 m glucose; 0.0002
mM TPN; 0.002 m PP; 25 y of hexokinase; and 250 y of glucose-6-phosphate dehydro-
genase. The reaction was started by the addition of 6.6 y of aceto-CoA-kinase
(specific activity 125).

reactions involved are as follows:

aceto-CoA

(9) Adenyl acetate + PP ———— ——> ATP + acetate
kinase
hexokinase >
(10) ATP + glucose ——_———_———  glucose-6-phosphate + ADI]

Mgtt+

glucose-6-phosphate

11 -6- N
(11) Glucose-6-phosphate + TP dehydrogenase

6-phosphogluconate + TPNH + Ht

By the spectrophotometric assay, the effect of adeny] acetate concentra-
tion on the initial rate of ATP formation (TPN reduction) was determined.
The maximal rate was obtained with 2 X 10-4 mM and the half maximal rate
at 5 < 10-'m. Ina similar experiment with adeny] acetate in excess and
P. BERG 1003

the PP concentration varied, half maximal rate was found between 1 and
5 X 10-5 M.

Mgt* Requirement—The conversion of adeny] acetate to ATP required
Mg*t+. By measuring the conversion of P?P® to ATP, a maximal rate was
found with 1.5 X 10-*m Mgt, a half maximal rate with 2.5 X 10-‘M, while
with no Mgt the rate was 2 per cent of maximum. According to the PP-
ATP exchange assay, the maximal rate of exchange occurred with 5 X 1073
M Mgtt. The discrepancy of the observed optimal concentration of Mgtt+
by the two methods of measurement may be due to the presence of relatively
larger amounts of ATP in the latter assay. Higher concentrations of Mg++
were Inhibitory; with 1.5 & 10°? Mm and 2.5 X 10-? M, the inhibition was 15
and 33 per cent, respectively. At these higher concentrations there was a

 

 

Taste VIII

Effect of CoA on PP-ATP Exchange and Conversion of Adenyl Acetate to ATP
Experiment No. Components P#P® incorporated into ATP

: umole

1 Complete 0.67

“ + 2 umoles CoA 0.14

2 Complete 0.23

“s + 2 umoles CoA 0.015

 

Experiment 1. A volume of 1.0 ml. contained 0.1 m potassium phosphate, pH
7.5; 0.005 m MgCl.; 0.002 ma ATP; 0.002 m P#2P3?; 0.001 m acetate; 0.01 m glutathione;
7.2 of enzyme (specific activity 96). Incubated at 37° for 20 minutes. Experiment
2. Same as above, except with 0.0003 m adenyl acetate instead of ATP.

visible precipitate (presumably some complex of Mg*t and one or more of
the phosphate components).

_ Conversion of Adenyl Acetate to Acetyl CoA—It was previously reported
that CoA inhibits the exchange of PP with ATP by aceto-CoA-kinase (20).
This has been confirmed by using the purified enzyme activated with ace-
tate. Moreover, the conversion of adenyl acetate to ATP is markedly in-
hibited by CoA (Table VIII). This inhibition is due to a competition for
the substrate, adenyl acetate, by CoA and PP, as demonstrated by the
rapid conversion of adenyl acetate to acetyl] CoA. Acetyl CoA formation
from adeny! acetate and CoA was measured spectrophotometrically at
340 my by coupling with the malic dehydrogenase-citrate-condensing en-
zymes (11, 29), as shown in Fig. 3. It can be seen that DPN reduction is
dependent on the presence of adeny] acetate, CoA, and aceto-CoA-kinase.
The increase in optical density at 340 mz in Fig. 3 (0.789) is equivalent to
the formation of 0.127 umole of acetyl CoA, which is in agreement with the
0.126 umole of adenyl acetate added.
1004 MECHANISM OF ACETATE ACTIVATION

With the malic dehydrogenase-citrate-condensing enzyme assay, the
effect of adeny] acetate concentration on the initial rate of acety! CoA for-
mation was studied. A maximal rate was obtained with 2 x 10-‘m anda
half maximal rate at 5 X 107° M.

L00

 

NOL

   

3

Complete

 
   
 

| Density at 340 mp
$8 3 8 8

Optical
8

No Aceto-CoA-kinase
—— 3S
No Condensing Enzyme

5 0 6% 2 2 30 35
Minutes

Fic. 3. Formation of acetyl CoA from adenyl acetate and CoA. Reaction mix-
ture: 1.0 ml. containing 0.1 m potassium phosphate, pH 7.5; 0.005 m MgCl.; 0.00015
Mm CoA; 0.005 st glutathione; 0.0005 m DPN; 0.005 m [-malate; 40 of crystalline citrate-
condensing enzyme; 18 y of aceto-CoA-kinase (specific activity 72); and 0.126 nmole
of adenyl acetate (assayed by conversion to ATP). Adeny] acetate was added ini-
tially to Curves 1, 2, and 3, and, at 6 minutes, to Curve 4. CoA was added to 3 at 23
minutes. The experiments were carried out at room temperature in cuvettes with
a lem. light path.

 

 

 

The acetyl CoA formed from adeny] acetate and CoA was further identi-
fied by its ability to acetylate p-nitroaniline with a partially purified amine-
acetylase from pigeon liver ((32); (Fig. 4)). The over-all stoichiometry of
the reaction could not be determined under these conditions, probably due
to the non-enzymatic acetylation of thioglycolate by acetyl CoA (32).

Mgt* Requirement for Conversion of Adenyl Acetate to Acetyl CoA—In
contrast to the ATP-PP exchange and the conversion of adeny! acetate to
ATP reactions, which have an absolute requirement for Mgt’, acetyl CoA
P. BERG 1005

formation occurs maximally in the absence of added Mgtt. Adeny] acetate
and CoA were incubated with the enzyme, and various concentrations of
Mgt+ and the acetyl CoA were measured as previously described. The
same amount of acetyl CoA was formed in the absence of added Mgt? as in
the presence of up to 5 X 107? m Mgt,

Attempts to Demonstrate Enzymatic Synthesis of Adenyl Acetate—The re-
actions of adenyl acetate demonstrated in the previous sections are in agree-
ment with those predicted by the hypothesis shown in Reactions 7 and 8.

600

 

o—@
S

re “A NoCoA J

730 /- No Aceto-CoA-kinase

Tl
Oo
oa

Oo
wn
So

900

500. Complete

Optical Density. at 420my
g
T

 

 

450 L | | L i t
5 io 8615020 2530
Minutes

Fig. 4. Acetylation of p-nitroaniline with adenyl acetate and CoA. Reaction
mixture: A volume of 1.0 ml. contained 0.1 m potassium phosphate, pH 7.6; 0.005
M MgCi,; 0.00013 M p-nitroaniline; 0.005 m ethylenediaminetetraacetate; 0.005 mu
thioglycolate; 0.00015 mu CoA; 0.003 m adenyl] acetate; 18 y of aceto-CoA-kinase (spe-
cific activity 72); 0.05 ml. of acetylating enzyme (acetone fraction, 1060 units per ml.
(32)). Adenyl acetate was added at zero time. The experiments were carried out at
room temperature in cuvettes with a1 cm. light path.

 

Attempts to achieve the net enzymatic synthesis and isolation of adenyl
acetate from ATP and acetate have been unsuccessful to date. By using
P*labeled ATP and an amount of crystalline inorganic pyrophosphatase
(33) sufficient to hydrolyze 150 umoles of PP per minute (200 ), there was
no significant increase in the P® of the non-nucleotide phosphate fraction
above a blank, with no acetate, under conditions in which a liberation of
0.005 umole of PP could have been detected.

In an attempt to trap adenyl acetate by an experiment in isotope dilution,
adenine-C4-labeled ATP, adenyl acetate, Mgt+, andthe enzyme were incu-
bated in the presence and the absence of acetate. After 30 minutes, the
mixture was adjusted to pH 10 to hydrolyze the adeny] acetate to A5P (22),
1006 MECHANISM OF ACETATE ACTIVATION

and then chromatographed on an anion exchange column (28). The results
(Table IX) show that there was no significant incorporation of the A5P
moiety of ATP into the adenyl acetate pool. Similar results were obtained
in the presence of 100 y of inorganic pyrophosphatase.

A rational explanation for the above failure of isotope equilibration be-
tween ATP-C" and adenyl acetate became apparent when it was found that
adeny] acetate inhibited the utilization of ATP. ATP, labeled with P® in
the terminal pyrophosphate group, was incubated with acetate, reduced
CoA, inorganic pyrophosphatase, and aceto-CoA-kinase in the presence and
the absence of adenyl acetate. After incubation for various periods of time,
aliquots were assayed for acetyl] CoA and the PP liberated was determined
in the supernatant fluid after adsorption of the ATP-P® with Norit. In

TasBLe IX
Attempted Trapping of Enzymatically Formed Adenyl Acetate

 

Specific activity

 

 

Components
ASP ATP
c.p.m. per umole C.p.m. per pmole
Complete................... 117 20,450
Minus acetate.............. 125 21,450
“  enzyme.............. 91 22,350

 

A volume of 1.0 ml. contained 0.10 M potassium phosphate, pH 7.5; 0.005 m MgCl;;
0.002 m ATP containing 2.5 X 104 c.p.m. per umole; 0.002 m acetate, 0.0055 m adenyl
acetate, and 36 y of enzyme (specific activity 72). Incubated 30 minutes at 37°.

the absence of adeny] acetate, P®?P*? liberation and acetyl CoA formation
were equal throughout the incubation (Fig. 5). In the presence of adenyl
acetate, however, there were a marked decrease in the PP formed and an
increase in the rate of acetyl CoA formation. With smaller amounts of
adeny] acetate, the inhibition of P**P"? formation was not as marked, and as
the adenyl acetate was utilized for acetyl CoA formation, the rate of P#?P®
formation increased. These data suggest that, in the presence of adenyl
acetate and ATP, there was a preferential use of the adenyl acetate. This
conclusion was supported by competitive inhibition experiments with PP
formation from ATP as a measure of ATP utilization. With ratios of ATP
to adeny] acetate of 1.7, 0.56, and 0.42, the inhibition was 68, 85, and 92 per
cent, respectively. In measuring the conversion of adenyl acetate to ATP,
a ratio of 8 of ATP to adeny] acetate produced only a 25 per cent inhibition.
From this it appears that the affinity of adeny] acetate for the enzyme is
greater than that of ATP. This finding, therefore, makes trapping experi-
ments with labeled ATP or acetate experimentally difficult.
P., BERG 1007

Exchange of A6P-C™ and AT P—According to the hypothesis proposed by
Jones et al. (20), aceto-CoA-kinase should catalyze an exchange of A5P with
the corresponding moiety of ATP in the presence of CoA alone (see Reac-
tions 4, 5, and 6). By the hypothesis proposed in Reactions 7 and 8, the
exchange requires both CoA and acetate. Adenine-C'*-labeled A5P was
incubated with unlabeled ATP and the enzyme in the presence or absence
of acetate and CoA, and then A5P and ATP were separated by anion ex-
change chromatography (28) and the radioactivity determined (Table X).

 

 

 

D.56L.
E
2 asl 2ymoles Ad-Ac
> tumole Ad-Ac
& 40
© 40,
3 -_
Sel ZA No Ad-Ac
= 24 4 ----PP
o | Vi __——Acety! Coa
a 4
ao
= 6 L Y
a t
.08 ¢ _~»!Umole Ad-AC
eT umoles Ad-AC
wpe" T” 4

 

I
Is 30 45 60
Minutes

Fic. 5, Effect of adeny!] acetate on utilization of ATP for acetyl CoA synthesis.
Reaction mixture: 1.0 ml., containing 0.05 m Tris, pH 7.5; 0.005 m MgCl,; 0.005 m
acetate; 0.001 m ATP?? (ARPP#P#) containing 43,500 c.p.m. per umole; 0.0007 m re-
duced CoA; 100 y of crystalline inorganic pyrophosphatase; 3.6 y of enzyme (specific
activity 72). Temperature 37°.

In the complete system during the time the exchange occurred, there was
0.32 umole of acetyl CoA formed, as measured by acethydroxamic acid.
These data demonstrate that there was an exchange of A5P and ATP only
under conditions in which complete reversal of the reaction occurs; namely,
when both acetate and CoA were present, and are in agreement with the
mechanism proposed in Reactions 7 and 8. Moreover, this experiment
supports the earlier conclusion that trace quantities of CoA are not present
in the enzyme preparation.

Exchange of Acetate-C'* with Acetyl CoA—Jones et al. (20) have reported
that aceto-CoA-kinase catalyzed an exchange of acetate-C" with the acetyl
moiety of acetyl CoA in the absence of A5P, PP, and Mgtt. Since this
observation was inconsistent with the mechanism proposed here, the ex-
1008 MECHANISM OF ACETATE ACTIVATION

change experiment was reexamined with the purified enzyme (Table XI).
In the absence of both A5P and PP, and with PP alone, there was only a
small amount of exchange detectable. When A5P alone was added, the in-
corporation of acetate-C" into acetyl CoA was about 30 per cent of the

TaBLe X
Exchange of A6P-C and ATP by Aceto-CoA-kinase

 

Specific activity

 

 

Components
ASP ATP
C.p.m, per pmole C.p.m. per umole
Complete................... 69,500 2430
Minus acetate.............. 113,000 0
CoA... eee. 118,000 | 0
“acetate and CoA..... 114,000 | 0

 

A volume of 1.0 ml. contained 0.1 m potassium phosphate, pH 7.5; 0.005 m MgCl;
0.0024 m ATP; 0.0069 m C4-AS5P, specific activity 1 X 105 c.p.m. per zmole; 0.0005 m
reduced CoA; 0.003 m acetate; 8.3 y of enzyme (specific activity 125). Temperature
37°; incubation, 30 minutes.

TaBLE XI
Exchange of Acetate-C'' and Acetyl CoA by Aceto-CoA-kinase

 

7 oe Specific activity Per cent
Experiment No. Additions of acetyl CoA exchange

 

C.pm,. per winole

l None 1,400 0.38
2 ABP 106 , 000 29.2
3 PP 1,950 0.54
4 A5P + PP 111,000 30.5
5 «+ PPase 0 | 0.00
6 eo ‘© + fluoride + PP 102,500 | 28.2

 

 

 

A volume of 0.27 ml. contained 0.15 m Tris, pH 7.4; 0.01 m MgCl.; 0.001 m ace-
tate-C'; 6.7 X 105 ¢.p.m. per umole; 0.00084 m acetyl CoA; 5.4 y of enzyme (specific
activity 128); and, where indicated, 0.0011 m A5P; 0.0019 m PP (Experiment 3);
0.007 m PP (Experiment 6); 50 y of inorganic pyrophosphatase; 0.093 M potassium
fluoride. Temperature 37°; incubation 30 minutes. Acetyl CoA was separated from
acetate-C'* by adsorption on Norit and elution with 50 per cent ethanol containing
0.005 «a KOH. Aliquots were counted and the acetyl CoA content determined as
acethydroxamic acid.
P. BERG 1009

calculated maximum under these conditions, and this was not increased by
adding PP. Since the exchange in the presence of A5P alone also required
Mgt, it appeared likely that this was due to small amounts of PP in the
reaction mixture. Addition of inorganic pyrophosphatase in the presence
of A5P completely blocked the incorporation of acetate-C" into acetyl CoA.
When, in addition to the inorganic pyrophosphatase, sufficient fluoride to
inhibit the pyrophosphatase and PP were added, the exchange was re-
stored. These data show, therefore, that both A5P and PP are required
for the acetate-acetyl CoA exchange and are in agreement with the mech-
anism postulated in Reactions 7 and 8. These experiments also suggest
that the acetyl moiety of adeny)] acetate does not exchange with free ace-
tate. This has been confirmed by incubating adenyl acetate and
acetate-C“ with the enzyme and reisolating the adenyl acetate. There
was no detectable incorporation of acetate-C™ into the recovered adenyl
acetate.

DISCUSSION

The formation of adeny] acetate as an intermediate in acetyl CoA forma-
tion by yeast aceto-CoA-kinase is supported by the following observations.
(1) The exchange of PP and ATP requires acetate; (2) synthetic adeny]
acetate is readily converted to ATP in the presence of PP and to acetyl-CoA
with CoA; (3) the CoA-independent accumulation of acethydroxamic acid,
PP, and A5P in the presence of hydroxylamine; (4) the requirement of ace-
tate and CoA for the exchange of A5P with ATP (5); and the requirement
of A5P and PP for the exchange of acetate with acetyl CoA. Further sup-
port for the proposed mechanism has been provided by the recent O" ex-
change studies of Boyer et al. (48). It was found that O'*-carboxyl-labeled
acetate gave rise to excess 08 in the phosphate group of A5P. These re-
sults would be predicted from Reactions 7 and 8 and are discussed below.

The failure to accumulate enzymatically formed adeny] acetate has been
puzzling. Whether this is a reflection of a low dissociation of adeny] ace-
tate from the enzyme is not clear. The apparent dissociation constant
(K,) of adenyl acetate is about 5 X 10-5 Mm, but whether this is the same as
the dissociation constant of enzymatically formed adenyl acetate is not
known. Further work is required to elucidate this point.

In considering the mechanism of the aceto-CoA-kinase reaction and its
analogy with the formation and utilization of acetyl phosphate, certain
similarities are apparent. Koshland (49), in a discussion of the mechanism
of group transfer reactions, pointed out that many of the well known en-
zymatic group transfers may be considered as single displacement or sub-
stitution reactions. With the phosphorylation of acetate to illustrate this,
3010 MECHANISM OF ACETATE ACTIVATION

the reaction may be formulated as follows:
1 O- O- O-

| | |
CH,C—O: + -O—Pt—O—P'—-0—P+—-O— adenosine —

o- O- o-
Oo O- O- O- O7-
| NZ | |
(12) CH,;C—O::-- o tee OTF OE O—adenosine->
0O- O- O-
oO O- O- Q-

I | |

CH;C—O—Pt—O- + OOO adenosine
Oo- O- o-

First, there is a nucleophilic attack on the terminal P atom by the un-
shared pair of electrons of the acyl oxygen atom, forming an activated com-
plex which breaks down to ADP and acetyl phosphate. In the case of the
aceto-CoA-kinase reaction, however, the nucleophilic attack by acetate
may be considered to occur in a similar way, but on the adenylic acid phos-
phorus atom liberating PP and forming a derivative of acetyl phosphate;
namely, adenyl acetate. The formation of acetyl CoA by phosphotrans-
acetylase and by aceto-CoA-kinase also appears to be analogous when con-
sidered in this way. Thus a nucleophilic attack by CoA on the acyl carbon
atom displaces, in one case phosphate, and in the other, A5P (Reaction 13).

Lo inne
CoA S: + if enesine —>+CoA S:--- i, --Q-—-P+—O—adenosine—>
H |
oO O- oO O-
(13)
o-

CoA S—C—CH, + ~O—Pt—O—adenosine

|

0 O-

Analogous reactions which lead to the liberation of PP are well known.
In the enzymatic formation of DPN (36), FAD (89), and UDPG (40), one
may consider the nucleophilic displacing unit to be the phosphate-oxygen
atom of NMN, FMN, and glucose-1-phosphate, and the acceptor, the A5P
moiety of ATP, or the U5P moiety of UTP.

Although it is clear that the formation of acetyl CoA via acety] phosphate
is catalyzed by two separate enzymes, all attempts to show that the aceto-
Pr. BERG 1011

CoA-kinase reaction involves more than one enzyme have been negative.
Thus far our own studies with the yeast enzyme have given no indication
of a separation of the activities shown in Reactions 7 and 8 during a purifica-
tion of the enzyme of about 50-fold, although further studies would be re-
quired to establish this point rigorously. Similar results have been
obtained with the acetyl CoA- and butyryl CoA-forming systems of animal
tissue (11, 19).

There is now plentiful evidence that the formation of acyl adenylate com-
pounds may be of considerable significance in other acy] group activations.
Thus, in the butyrate-activating system (19), the utilization of synthetic
adenyl butyrate for ATP and butyryl CoA formation has been demon-
strated. Furthermore, Jencks (50) has reported a CoA-independent for-
mation of octanoyl hydroxamic acid and PP by a pig liver enzyme, and it
appears possible that this represents an initial formation of adenyl octanoate
which is subsequently split by hydroxylamine.

During the course of this work (21) an enzyme was purified (23) from
yeast which catalyzes a PP-ATP exchange requiring specifically L-methi-
onine, and which in the presence of hydroxylamine carries out the following
reaction.

(14) ATP + u-methionine + hydroxylamine >
Lu-methionine hydroxamate + A5P + PP

Hoagland et al. (51) have also presented evidence for the formation of amino
acid hydroxamates and PP from ATP, and some fifteen amino acids by a
soluble protein fraction of liver. A similar system which requires amino
acids for an exchange of PP and ATP has been demonstrated in extracts of a
number of microorganisms (52). Maas (53), in his studies of pantothenic
acid synthesis by an enzyme from Escherichia coli, has shown that pantoic
acid is required for the exchange of PP with ATP. Because of this and
other evidence, Maas has suggested (53) the formation of a pantoyl adenyl-
ate compound which is subsequently cleaved by §-alanine, forming panto-
thenic acid. It is interesting to note that the nitrogen atom of
amino groups contains an unshared pair of electrons and is capable of initi-
ating a nucleophilic displacement of the A5P in the manner proposed for
the sulfur atom of CoA. Evidence has also been presented for the forma-
tion of adenyl sulfate from ATP and sulfate in the esterification of nitro-
phenol by liver enzymes (54) and for the formation of adenyl carbonate in
the carboxylation of 8-hydroxyisovaleryl CoA (55).

It therefore seems possible that acyl adenylate formation may represent
a general mechanism for the activation of fatty acids, amino acids, and a
variety of compounds containing an acyl group.

3 Private communication from Dr. H. Beinert and Dr. C. N. Lee Peng.
1012 MECHANISM OF ACETATE ACTIVATION

SUMMARY

Studies of the enzymatic mechanism of acetyl CoA synthesis from ATP,
acetate, and CoA have shown that the reaction occurs in two steps.

ATP + acetate < adeny! acetate + PP

Adenyl-acetate + CoA = acetyl CoA + A5P
ATP + acetate + CoA = acetyl CoA + ASP + PP

 

In support of this formulation are the following observations: (1) The ex-
change of PP? with ATP requires acetate; (2) acethydroxamic acid is
formed from ATP, acetate, and hydroxylamine in the absence of added
CoA; (3) synthetic adenyl acetate is converted to ATP in the presence of
PP and to acetyl CoA in the presence of CoA; (4) the exchange of A5P-C%
with ATP is dependent on the presence of acetate and CoA; and (5) the
exchange of acetate-C" with acetyl CoA requires both A5P and PP.

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