ACYL ADENYLATES: AN ENZYMATIC MECHANISM
       OF ACETATE ACTIVATION*

             BY PAUL BERGt
   WITH THE TECHNICAL ASSI~TANCE OF GEORGIA NEWTON
(From the Department of Microbiology, Washington University School of
         Medicine, St. Louis, Missotrri)

      meceived 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 phosphoryIation of acetate with ATP1 by acetokinase (3, 4),
followed by the transfer of the acetyl group to CoA by the action of phos-
photransacetylase (5-7).

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

In animal tissues (8-11), yeast (12, 13), plants (14), and Rhodospirihm
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 Ft 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 acetyl group of
acetyl CoA in the absence of A5P and PP.  To account for these observa-
tions, the following mechanism was proposed (20).

and the National Science Foundation.

* This work was supported by grants from the United States Public Health Service

t Scholar in Cancer Research of the American Cancer Society.

1 The following abbreviations have been used: adenosine triphosphate, ATP;
adenosine-5'-phosphate , A5P; 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-!j'-phosphate, U5P.

991

992 MECHANISM OF ACETATE ACTIVATIOX

(4)

(6)

(6)

  ATP + enzyme e enzyme - A5P + PP
Enzyme - A5P + CoA e enzyme - CoA + A6P
Enzyme - CoA + acetate 2 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).

iOJ

0- 0

0-

Adenyl acetate has been synthesized and demonstrated to react enzymat-

ically in the manner shown below.

(7)

(8) Adenyl acetate + CoA acetyl CoA + A5P

A preliminary communication of this work has been reported elsewhere (21).
The synthesis, purification, and characterization of adenyl acetate are de-
scribed in the following paper (22).  The apparently analogous formation
of an amino acid acyl adenylate in the reaction of L-methionine with ATP
by an enzyme from yeast has been briefly ment'ioned earlier (21) and will be
reported in detail in an accompanying paper (23).

ATP + acetate e adenyl acetate + PP

Afaterials and Methods

P32P32 was prepared by heating Na2HP3204 at 225" for 18 hours (241, and
purified by anion exchange chromatography.  ATP, labeled with P32 in the

P. BEIZG 993

terminal pyrophosphate group, was prepared either by exchange of P32P32
with unlabeled ATP by using purified aceto-CoA-kinase (Reaction 7) or
from A5P and P3? by rat liver mitochondria in the presence of cr-ketoglu-
tarate. Adenine-C14-labeled ATP was prepared by first coupling ad-
enine-C1* with 5-phosphoribosyl pyrophosphate (25) and converting the
A5P-C14 to ATP-C14 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 C1- column and eluting
with 0.1 N HC1-0.1 M KC1.  According to the opbical density at 260 nip
and acetyl CoA assay (11, 29), it was 50 per cent pure. Sodium
acetate-l-C1* was obtained from Tracerlab, Inc., and sodium fluoroacetate
was kindly supplied by Dr. R. 0. 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 et al. (13).

Hexokinase was prepared by a modification of the method of Brown? and
contained 5 units per mg. (1 unit forms 1 pmole 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. S. 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 aE. (32).  Crystalline inorganic 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

2 We are grateful to Dr. D. H. Brown for giving us this method before its publica-
tion.

994 MECHANISM OF ACETATE ACTIVATIOh-

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

Acetyl co.4 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 COG, measured as mentioned above, or by conversion of P32P32 to
ATP (see Table VI).  Adenyl 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 et al. (13).  The reaction mixture contained, in 1.0 ml.,
0.1 M potassium phosphate, p1-I 7.5, 0.005 nr MgC12, 0.01 M ATP, 0.05 xi
potassium fluoride, 0.01 M glutathione, 0.15 nig. of CoA (equivalent to 0.1
pmole), 0.01 M potassium acetate, 0.2 M neutralized hydroxylamine, and the
enzyme.  The mixture was incubated for 20 minutes at 37", then 2 nil. of
an acidified ferric chloride solution (13) were added.  After centrifugation
to remove precipitated protein, the optical density at 540 mp was deter-
mined with a Beckman model DU spectrophotometer.  In all cases, a con-
trol tube in which CoA WM omitted was incubated with each amount of
enzyme.  The formation of 1 pmole 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 prnole of acethydros-
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).

Ezchunge of P32P32 with ATP-The standard assay mixture contained, in
1.0 ml., 0.1 M potassium phosphate, pH 7.5, 0.005 M MgC12, 0.002 M ATP,
0.001 M acetate, 0.05 M fluoride, 0.002 M P32P32 containing between lo4 and
los c.p.m. per prnole, 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, follon-ed by 0.2 nil. of a suspension of acid-washed Xorit (15 per
cent, dry weight).  After 3 minutes, the Korit 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 P32 content determined with a Geiger-Muller
counter.  The activity is expressed as the micromoles of P32P32 incorpo-
rated into ATP and is calcuiated by dividing the total P32 activity in ATP
by the specific activity of the initial P32P32.  1 unit of enzyme activity is
defined as the incorporation of 1 pmole of P32P32 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

PuriJication 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 kc. sonic oscillator (Ray-
theon) for 40 minutes.  The mixture was centrifuged in a Serval1 centrifuge
at 10,OOO 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,OOO 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 ACTNATXON

8820
5875
3835

Oy 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 (Cr gel eluate).
To the Cr 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

21.4
2.0
1.9

25 and 35 per cent.

Fraction

Crude extract. ......
Protamine eluate. ...
AS-1 ................
Cy gel eluate.. ......
AS-2. ...............

Fraction AS-2 has been repeatediy frozen and thawed

       TABLE I
Purification of Aceto-CoA-kinase

Units
per ml.

49
47
59
35
157

SP+C*

rctlv1ty

vnils per mg.

protein
2.3
24
31

* Assay, formation of acetyl CoA, measured by formation of acethydroxamic acid

~EJ described in "Methods."

several times a week for periods of up to 4 months without appreciable loss
of activity .
P32P32 Exchange with ATP-It had been reported previously (20) that
aceto-CoA-kinase catalyzed an exchange of Pa2P3* 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, P32P32, and Mg++, acetate was required for the exchange reaction with
the most purified fraction of aceto-CoA-kinase (Table 11).  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 111.  Since the rdle of acetate is catalytic, small amounts of acetate

P. BERG 997

effect an appreciable amount of exchange of P3?P3* 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   M and higher has not been observed with acetyl CoA formation as

          TABLE I1
Requirements for PJ2PS' Ezchange with A TP

Components

Complete ...............................
Minus ATP. ............................
. " Mg*. ............................

acetate

enzyme.

ii

...........................

ii

.........................

P*Pu incorporated into ATP

/UMk
0.55
0.01
0.01
0.02
0.00

UsuaI assay procedure for PP-ATP exchange with 0.002 M acetate and 7.5 y of

enzyme (Fraction AS-2); specific activity 92.

             TABLE I11
Effect of Acetate Concentration on Pa'Par-ATP Ezchange

Concentration PnP* incorporated into ATP

I

X 10' n
0.0
0.4
2.0
10.0
30.0
40.0

pWlU
0.02
0.15
0.39
0.57
0.43
0.37

The conditions are the same tu for the experiment described in Table 11.

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 to loA6 M.

Time-Course and Extent of Exchange-The exchange of P32P32 and ATP
approaches the calculated value for complete equilibration of the pyrophos-
phoryl group of ATP and the PazP3? (Fig. 1).  ATP prepared in this way
contains essentially equal amounts of P32 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 ACTIVATIOIU'

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.        M, the rate was

At 5 X

FIG. 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 MgCl2; 0.0016 M
ATP; 0.001 M potassium acetate; 0.002 M PJ*Ps* containing 5 X lo4 c.p.m.; 4 -y of en-
zyme, specific activity 96,  Temperature 37".

about 30 per cent that found with 1 X    M acetate. With fluoroacetate
at levels of 1 X           M, there was about 2 and 5 per cent
the exchange found with 1 X    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 GOA synthesis was a reversible enzymatic interaction
of ATP and acetate with the formation of adenyl acetate and PP (Reac-

M and 5 X

P. BERG 999

Complete.. . . . . . . .

XIinus ncetirte. . . .

    enzynic.. . ,
    ATP.. . . . . .

'i

"

tion 7).  This hypothesis was tested with hydroxylamine to trap the acetyl
group of adenyl acetate its acethydrosamic acid.  With large amounts of
enzyme, ATP, and acetate, arid high concentrations of hydroxylamine
(2.5 M), there was a net formation of acethydroxatnic acid (Table IV), which
increased with time and \vas dependent on the presence of ATP, acetate,
and the enzyme.  When a smaller amount of hydroxylaniine was used,
(1.9. 1 M, there was only 16 per cent as much acethydrosamate formed.  The
rate of acethydroxamic acid formation was also dependent on the amount
of enzyme; thus, with 33, 82, lG4, and 330 y of enzynie, there was a forma-

pnwlc A 1 rmok pmolcs A

0.22 ! 0.20 , 0.22 ~

        0.04 j

1 1.38

0.52 I 0.30 I 0.89 ~

0.04 i 0.03

~ 0.04 1

I

0.04

                    TABLE IV
Fornialion o/ Acelhydtozuniic Acid from ATP and rlcetule in Absence of CoA

._ ~ .~ __ _____

Time

Components

60 min. 120 min.

I

I

30 min.

I

All the values are exyrcsscd in micronioles of ucethydroxamic acid niid the change
(A) waa calculated from the difference between the values obtained in the presence
and the absence of acetate.  Reaction mixture: 1.0 mi. contained 0.1 M potassium
phosphate, pH 7.5; 0.005 hi MgCI2; 0.01 M ATP; 0.01 hi 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 pmole of acethydroxamic acid in 60 min-
utes.
Ace t hy droxaniic 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 pmoles of acethydrosamic acid and 1.3 pmoles of A5P were formed,
and, in the absence of acetate, 0.1 pmole of acethydroxamic acid and 0.1
pmole of A5P were found.

The above results are in agreement with the postulated formation of
adenyl acetate ~ts an intermediate, an interpretation, however, dependent
on the assumption that small amounts of CoA were not present.  In order
to determine whether Coh 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 MECH:\NISbl OF ACET.ATF: ACTIV:\TIOK

pmolc

Complete.. . . . . . , .   0.74
Minus acetate.. . 0.09

enzyme . . . 0.03

1I

volume of only 1.5 ml., as little as 0.0005 pmole of CoA could be detected.
Heat-inactivated samples of the enzyme (330 r) 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.  As a control, CoA added
to the enzyme solution before or after heating was quantitatively recovered.
Moreover, when a sample of heated enzyme (330 7) 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 enxynic
alone, whereas the ddition of O.OOO.5 pmole of CoA doubled the formation
of acethydroxamic acid.  These experiment>s indicate that the acethy-

~- ~-

       amoles            pnrolcs
              0.76     1.12 I 0.73
0"; ~ i::: 0.10   0.46 1 0.07

0.35 0.39

            TABLE V
Stoichiometry of Products Formed in ATP-Acclatc
  Reaction in Presence of Hydroxgla?nine

Components

PP'

A

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 esperiments
discussed thus far, it was considered likely that the intermediate formed
from ATP and acetate was the phosphoacetyl derivative of A5P.  This
compound was synthesizcd first from the silver salt of A5P and acetyl 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 I1 (22).

P. BEHG 1001

Synthetic adenyl acetate was converted to Kl'Y in the presence of PP,
                       This was shown with P3*P3* and

Mg++, and aceto-CoA-kinase (Table VI).

           TABLE VI
Formation oj ATP from Adenyl ricetale and PP

i Time 1 PP incorporated into ATP

i

Cornpnen ts

Complete ...................

Minus enzyme.. ......
Complete*. ................

<(

...................

__

.. __

t?l in.

20
45
20
20

~

rmolc
0.23
0.28
0.01
0.00

* The adenyl 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 hi MgCI?; 0.002 hf P321)3*;
0.28 rmole of adenyl acetate (measured by hydroxamic acid assay); and 7.2 y of
enzyme (specific activity 72).

.36

1

FIQ. 2. Correspondence of ATP formation and adenyl acetate disappearance.
Reaction mixture: 1.0 ml. containing 0.1 M potassium phosphate buffer, pH 7.5;
0.005 M MgC12; 0.00032 M adenyl acetate; 0.002 M P3*PJ*; and 7.2 y of enzyme (specific
activity 72). Temperature37".  ATP determined by Norit assay and adenyl acetate
by hydroxamic acid assay.

by measuring P3? in ATP after adsorption and elution from Sorit.  iiccord-
ing to these data, ATP was formed in an amount equivalent to the amount
of adenyl acetate added.  Treatment of adenyl acetate 1vit.h 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 MECH.4X;ISM OF ACET.%TE -4CTIVATION

Adenyl acetate added*

pmolc
O.Oo0
0.024
0.037

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

The enzymatic conversion of adenyl acetate to A'l71' has also been verified
spectrophotometrically by being coupled to the phosphorylation of glucosc
with hexokinase.  Incubation of adenyl acetate, PP, big++, 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 1711).  The

TPNH formed

smolct

O.OO0
0.025, 0.025, 0.027
0.039, 0.040, 0.035

                    TABLE VI1
Formation of ATP from Adenyl Acetale and PP Measured Spectrophotontetrically
     with Hexokinase and Glucose-6-phosphate Dehydrogenase

* The adenyl acetate concentration was determined by acethydroxamic acid for-
mation and by conversion to acetyl CoA as measured with the malic dchydrogenaae-
citrate-condensing enzymes (10, a).
t An extinction coefficient of 6.22 X lo3 cni.-Lar-l (47) was uscd.  Reaction mix-
ture contained, in 1.0 ml., 0.05 hi Tris, pH 7.4; 0.001 M MgC12; 0.025 M glucose; 0.0o02
M TPN; 0.002 hi 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:

nceto-CoA
kinase

Adenyl acetate + PP

+ ATP + wetute

hexokinase
hlg++

ATP + glucose

> glucose-6.phosphate + A4111'

glucose-6-phosphate
dehydrogenase

(1 1) Glucose-6-phosphate + TPN

6-phosphogluconate + TPXH + H+

By the spectrophotometric assay, the effect of adenyl acetate concentra-
tion on the initial rate of ATP formation (TPN reduction) was determined.
The maximal rate was obtained with 2 X lod4 M and the half maximal rate
at 5 X 10-6 M.  In a similar experiment with adenyl acetate in excess and

P. BERG 1003

the PP concentration varied, half maximal rate was found between 1 and

Mg++ Requirement-The conversion of adenyl acetate to ATP required
Mg++.  By measuring the conversion of P32P32 to ATP, a maximal rate was
found with 1.5 X   M Mg++, a half maximal rate with 2.5 X   M, while
with no Mg++ the rate was 2 per cent of maximum.  According to the PP-
ATP exchange assay, the maximal rate of exchange occurred with 5 X low3
nr Mg++.  The discrepancy of the observed opt,inial concentration of Mg++
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 x lo-* M and 2.5 X   M, the inhibition was 15
and 33 per cent, respectively.  At these higher concentrations there was a

5 x M.

                   TABLE VI11
Eflect of CoA on PP-ATP Etchange and Conversion of Adenyl Acetate to ATP

Components

1 PalP*l incorporated into ATP

I

Experiment No.

1

2

Complete

Complete

ii + 2pmoles CoA

" + 2 pmoles CoA

@mole

0.67
0.14
0.23
0.015

Experiment 1.  A volume of 1.0 ml. contained 0.1 M potassium phosphate, pH
7.5; 0.005 M MgC12; 0.002 M ATP; 0.002 M P39PJ2; 0.001 M acetate; 0.01 nr glutathione;
7.2 y 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++ and one or more of
the phosphate components).
Conversion of Adenyl Acetate lo 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 VIJI).  This inhibition is due to a compet.ition 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 adenyl acetate and CoA was measured spectrophotometrically at
340 mp 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 adenyl acetate, CoA, and aceto-CoA-kinase.
The increase in optical density at 340 mp in Fig. 3 (0.789) is equivalent to
the formation of 0.127 pmole of acetyl CoA, which is in agreement with the
0.126 pmole of adenyl acetate added.

loo4 MECHANXSM OF ACEXATE ACTIVATION

With the malic dehydrogenase-citrate-condensing enxynie assay, the
effect of adenyl acetate concentration on the initial rate of acetyl CoA for-
mation was studied.  A maximal rate was obtained with 2 X loy4 b and a
half maximal rate at 5 X

M.

5 IO 15 20 25 30 35

Minutes

FIG. 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 MgCl2; 0.00015
M CoA; 0.005 M glutathione;O.0005 M DPN;O.005 M Z-rnalate;40~ of crystallinecitrate-
condensing enzyme; 18 y of aceto-CoA-kinase (specific activity 72); and 0.126 pmoIe
of adenyl acetate (assayed by conversion to ATP).  Adenyl acetate was added ini-
tially to Curves I, 2, and 3, and, at 6 minutes, to Curve 4.  CoA waa added to 3 at 23
minutes. The experiments were carried out at room temperature in cuvettes with
a 1 cm. light path,

The acetyl CoA formed from adenyl 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).

Mg" Requirement for Conversion of Adenyl Acetate to Acetyl CoA-In
contrast to the ATP-PP exchange and the conversion of adenyl acetate to
ATP reactions, which have an absolute requirement for Mg*, acetyl CoA

P. BERG 1005

formation occurs maximally in the absence of added Mg++.  Adenyl acetate
and CoA were incubated with the enzyme, and various concentrations of
Mg++ and the acetyl CoA were measured as previously described.  The
same amount of acetyl CoA was formed in the absence of added Mg++ as in
the presence of up to 5 X

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.

M Mg++.

.800,

2750

F

.700

.650

,600

,550

.-

n

450 1 I I I 1 I I

5 IO 15 20 25 30

Minutes

FIU. 4. Acetylation of p-nitroaniline with adenyl acetate and CoA.

                                             Itenction
mixture: A volume of 1.0 ml. contained 0.1 M potassium phosphate, pH 7.6; 0.005
M MgCln; O.WO13 M p-nitroaniline; 0.005 M ethylenediaminetetraacetate; 0.005 M
thiogiycolate; 0.00015 M CoA; 0.003 M adenyl acetate; 18 y of aceto-CoA-kinase (spe-
cific activity 72); 0.05 ml. of acetylating enzyme (metone fraction, 1060 units per ml.
(32)).  Adenyl acetate wm added at zero time.  The experiments were carried out at
room temperature in cuvettes with n 1 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
P3*-labeled ATP and an amount of crystalline inorganic pyrophosphatase
(33) sufficient to hydrolyze 150 pmoles of PP per minute (200 y), there was
no significant increase in the Pa2 of the non-nucleotide phosphate fraction
above a blank, with no acetate, under conditions in which a 1iberat.ion of
0.005 pmole of PP could have been detected.

In an attempt to trap adenyl acetate by an experiment in isotope dilution,
adenine-C14-labeled ATP, adenyl acetate, Mg++, and the 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 adenyl 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-CL4 and adenyl acetate became apparent when it was found that
adenyl acetate inhibited the utilization of ATP.  ATP, labeled with P32 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-P3* with Norit.  In

               TABLE IX
A ttempled Trapping of Enzgmatically Formed Adenyl Acetate

SpccSc activity

Components

ATP

I

ASP

Complete. ..................
Minus acetate. .............
I< enzyme. .............

6.p.m. per pmolc

117
125
91

c.p.m. jnfimolc
20,450
21,450
22,350

A volume of 1.0 ml. contained 0.10 M potassium phosphate, pH 7.5; 0.005 M MgC12;
0.002 M ATP containing 2.5 X lo4 c.p.m. per rmole; 0.002 M acetate, 0.0055 M adenyl
acetate, and 36 7 of enzyme (specific activity 72).

Incubated 30 minutes at 37".

the absence of adenyl acetate, P32P32 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 P32P32 formed and an
increase in the rate of acetyl GOA formation.  With smaller amounts of
adenyl acetate, the inhibition of P32P32 formation was not as marked, and as
the adenyl acetate was utilized for acetyl CoA formation, the rate of P32P32
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 adenyl 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 adenyl acetate produced only a 25 per cent inhibition.
From this it appears that the affinity of adenyl 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 A&P-C14 and A TP-According to the hypothesis proposed by
Jones et ai. (ZO), aceto-CoA-kinase should catalyze an exchange of A5P with
the corresponding moiety of ATP in t.he 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-CL4-1abeled h5P 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).

a

a

40

ID

-Acetyl CoA

&.I urnole A6Ac
,,~2~rnoles Ad-Ac

I

15 30 45 60

Minutes

FIG. 5. Effect of adenyl acetate on utilization of ATP for acetyl Co.4 synthesis.
Reaction mixture: 1.0 ml., containing 0.05 M Tris, pH 7.5; 0.005 M MgC12; 0.005 hi
acetate; 0.001 M ATP (ARPP**Paz) containing43,MO c.p.m. per pmole; O.OOO7 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 \vas
0.32 pmole of acetyl GOA 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 co.4 are not present
in the enzyme preparation.

Exchange of Acetate-C14 with Acetyl CoA-Jones et al. (20) have reported
that aceto-Co-4-kinase catalyzed an exchange of acetate-CL4 with the acetyl
moiety of acetyl Coh in the absencc of A5P, PI', and Mg++,  Since this
observation was inconsistent with the mechanism proposed here, the ex-

1008 MECHANISM OF ACETATE -4CTIVATION

   A5P

C.#.NL per pmde

69,500

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-CI4 into acetyl CoA was about 30 per cent of the

   ATP

c.p.m. per pmolt

.-

2430

             TABLE X
Exchange of A6P-CL4 and ATP by Aceto-6'0.4-kinase

Specific activity
of acetyl CoA

c.p.m. per pinolc

1,400
106,000
1,950
111,Ooo
   0

Components

Per cent
exchange

0.38

0.54

0.00

29.2

30.5

Complete. ..................

Minus acetate. .............

" acetate and CoA.. ...

CoA

dL

.................

Specific activity

A volume of 1.0 ml. contained 0.1 M potassium phosphate, pH 7.5; 0.005 M MgCI?;
0.0024 AI ATP; 0.0069 M C14-A5P, specific activity 1 X lo6 c.p.m. per pmole; 0.0005 AI
reduced CoA; 0.003 A~ ncetnte; 8.3 y of enzyme (specific activity 125).  Temperature
37'; incubation, 30 minutes.

               TABLE XI
Ezchange of Acetate-C14 and Acetyl CoA by Aceto-Con-kinase

Experiment No.

Additions

None
A5P
PI'
A5P + PP

+ PPase

(' + 'L + fluoride + PP

A volume of 0.27 ml. contained 0.15 hi Tris, pH 7.4; 0.01 M MgC12; 0.001 M ace-
tate-C"; 6.7 X 106 c.p.m. per pmole; 0.00084 hi acetyl CoA; 5.4 y of enzyme (specific
activity 128); and, where indicated, 0.0011 nl A5P; 0.0019 M PP (Experiment 3);
0.007 M PP (Experiment 6); 50 y of inorganic pyrophosphatase; 0.093 M potassium
fluoride.                       Acetyl CoA was separated from
acetate-C'4 by adsorption on Norit and elution with 50 per cent ethanol containing
0.005 M KOH. Aliquots were counted and the acetyl CoA content determined as
ncethydroxamic acid.

Temperature 37"; incubation 30 minutes.

1'. 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
Mg*, 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-C14 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 adenyl acetate does not exchange with free ace-
tate. This has been confirmed by incubating adenyl acetate and
acetate-C14 with the enzyme and reisolating the adenyl acetate.  There
was no detectable incorporation of a~etate-C~~ into the recovered adenyl
acetate.

DISCUSSION

The formation of adenyl acetate as an intermediate in acetyl CoA forrna-
tion by yeast aceto-CoA-kinase is supported by the following observations.
(1) The exchange of PP and ATP requires acetate; (2) synthetic adenyl
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 0l8 ex-
change studies of Boyer et al. (48).  It was found that O1*-csrboxyl-labeled
acetate gave rise to excess OI8 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 adenyl acetate has been
puzzling.  Whether this is a reflection of a low dissociation of adenyl ace-
tate from the enzyme is not clear.  The apparent dissociation constant
(K,) of adenyl acetate is about 5 X   M, but whether this is the same as
the dissociation constant of enzymatically formed adenyl acetate is not
known.

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,

Further work is required to elucidate this point.

I010 MECHANISM OF ACETATE ACTIVATION

the reaction may be formulated as follows:

0 0- 0- 0-

II I I I
             1
      I

CHaC-0: + -O--pf-+P+---pf-+adenosine -+

                  0-

0-

I

0-

0 0- 0- 0- 0-

II \/ I 1
            I

(12)

CHIC-: - - - SI'+. - - - O--Pt-+l'+-+adenosine-,

                 0-

0-

I

I

0-

0 0- 0- 0-

II I I I
             I

CHIC-el?--- + -O-P+-O-P+-O-adenosine

0-

I

0-

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-Cob-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).

1

0-

CH, 0- CHs 0-

I I I I
                  I

CoA 6: + C+P+Uadenosine --+ CoA S: ...C. - .&l?--0-adenosine-+

(13)

0-

II

0

I

0-

H II

0

0-

I

CoA S-C-CHI + -0-P+-0-adenosine

I

0-

11

0

Analogous reactions which lead to the liberation of PP are well known.
In the enzymatic formation of DPN (36), FAD (39), and UDPG (40), one
may consider the nucleophlic 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 acetyl phosphate
is catalyzed by two separate enzymes, all attempts to show that the aceto-

P. BERG 101 1

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 acyl group activations.
Thus, in the butyrate-activating system (19), the utilization of synthetic
adenyl butyrate for ATP and butyryl CoA formation has been demon-
~trated.~  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 + L-methionine + hydroxylamiue +

L-methionine hydroxamate + A5P + PP

Hoagland et d, (51) have also presented evidence for the formation of amino
acid hydroxamates and PP from ATP, and some fifteen amino acids by zl
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 @-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 Yeng.

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 $ adenyl acetate + PP

Adenyl-acetate + CoA ;=r acetyl CoA + A5P
ATP i- acetate + CoA e acetyl CoA + A5P + PP

In support of this formulation are the following observations: (1) The es-
change of P12Paz 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-ClJ
with ATP is dependent on the presence of acetate and CoA; and (5) the
exchange of acetate-C14 with acetyl CoA requires both A5P and PP.

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