Fatty acid biosynthesis. VIII. The fate of malonyl-CoA in fatty acid biosynthesis by purified enzymes from lactating-rabbit mammary gland

BIOCHIMICA

FATTY VIII.

ET BIOPHYSICA

ACTA

391

ACID BIOSYNTHESIS

THE

FATE

BY PURIFIED

OF MALONYL-CoA

ENZYMES

IN FATTY

ACID BIOSYNTHESIS

FROM LACTATING-RABBIT

MAMMARY

GLAND

H.

J. M. HANSEN*,

E. M. CAREY

AXD

R.

DILS

Department of B~~c~e~~st~y, The Medical School, ~l~~ve~s~tyof hl~~~~ngh~~, No~~~ngha~ h’G7 zRD

(GreatR&zin)

(Received July

gth,

19711

SUMMARY

r. We have investigated the formation and utilization of malonyl-CoA in fatty acid synthesis catalysed by preparations of partially purified acetyl-CoA carboxylase and purified fatty acid synthetase

from Lactating-rabbit

mammary

gland.

Carboxylation of [I-%]acetyl-CoA was linked to fatty acid synthesis by the presence of fatty acid synthetase and NADPH. The rate of fatty acid formation was equal to that of acetyl-CoA carboxylation, without the accumulation of free malonyl-CoA to a concentration required to obtain the same rate of fatty acid synthesis from added [r,3-l*C,]malonyl-CoA. 3. The preparations of acetyl-CoA carboxylase and fatty acid synthetase were each able to decarboxylate [r,3-14C,]malonyl-CoA. 4. Both enzyme preparations acted as competitive inhibitors of X0, fixation into acetyl-CoA catalysed by acetyl-CoA carboxylase in the absence of NADPH. This is attributed to a mechanism of product inhibition. The effect on the apparent activity of acetyl-CoA carboxylase assayed by malonyl-CoA formation is discussed. 5. Our results suggest a metabolic compartmentation of the carboxylation step which facilitates the incorporation of carboxylated acetyl-CoA into fatty acids in the presence of NADPH and prevents its catabolism by side reactions. 2.

INTRODUCTION

The biosynthesis of fatty acids involves a sequence of enzymic reactions. Acetyl-CoA carboxylase (EC 6.4.1.2) produces the substrate malonyl-CoA which is utilized by fatty acid synthetase. The question arises to what degree the malonyl* Address (for reprints) : Atomic Energy Commission Research Establishment, R&ii, 4000 Roskilde,

Denmark.

Biochim. Biophys. Acta, 248 (1971) 3gI-405

H. J. 111.HANSEN

392

et al.

CoA pathway is based on a “free” intermediate malonyl-CoA pool. Previous work1,2, * using purified enzymes from lactating-rabbit mammary gland and rat liver has shown an apparent lack of malonyl-CoA accumulation when the carboxylation of acetylCoA is linked in sitzt to fatty acid synthesis by the presence of fatty acid synthetase and NADPH. It appeared that at low concns. of substrate and of acetyl-CoA carboxylase the carboxylated acetyl-CoA had been completely incorporated into fatty acids. In the present investigation we have studied the enzymic formation of malonylCoA from [I-i*C]acetyl-CoA both in the presence and absence of fatty acid synthetase, and both with and without linkage to fatty acid synthesis via added NADPH. We have compared these results with others obtained by adding [I,3-*4C,]malonyl-CoA to fatty acid synthetase in a series of corresponding incubations. Ion-exchange chromatography has been used to analyse the products formed during these reactions. Only a small malony-CoA pool accumulated in the reactions which were linked to fatty acid synthesis. In the non-linked systems, side reactions occurred to a substantial degree. The effects of these side reactions on the assay of acetyl-CoA carboxylase by X0, fixation has been investigated. MATERIALS

The animals and most of the materials used have been described3. Sepharose 4B and Sephadex G-IO were obtained from Pharmacia (G.B.) Ltd., London W. 5; and Sagavac 4B from Seravac Laboratories Ltd., Moneyrow Green, Holyport, Maidenhead, England. DEAE-23 cellulose was purchased from Whatman Ltd., London E.C. 4; and catalase (EC 1.x.1.6) from Sigma Chemical Co., London. jr-14C]acetvl-CoA (specific activitv, $3 ~C~~rnole) and [~,3-l*C~~malonyl-CoA (specific activity, IO pC/pmole) were obtained from New England Nuclear Chemicals, Dreieichenhain, W. Germany. The latter substrate was purified on a DEAE-23 cellulose column as described in METHODS (Identi&ation of reactiofa products). Fractions containing [r,3-i3C,]malonyl-CoA were pooled and lyophilised. The residue was dissolved in 2.0 ml of water and desalted by chromatography on a column of Sephadex G-IO (20 cm x I cm). METHODS

Enzynze units Enzyme activity units are expressed as nmole X0, incorporated into acidstable material per min for acetyl-CoA carboxylase, and nmole malonyl-CoA incorporated into long-chain fatty acids per min for fatty acid synthetase at 37”. Partial

$uriJication

of acetyl-CoA

carboxylase

from

lactating-rabbit

mammary

gland

Acetyl-CoA carboxylase was partially purified by a method which combined some of the features used in the purification of the enzyme from chicken liver* and rat mammary gland&. Acetyl-CoA carboxylase is present in both the microsomal and particle-free supernatant fractions of homogenates of lactating-rabbit mammary *

Refs. I and 2 are parts VI and VII in this series, respectively.

Biochirn. Biophys.

A&z,

248 (1971) 39x-405

393

FATTY ACID BIOSYNTHESIS. VIII

glands. Consequentlyafterhomogenisationof the glands6 atroom temperature in 5omM potassium phosphate buffer (pH 7.0) containing 20 mM potassium citrate, I mM dithiothreitol, 0.1 mM EDTA and ~0% (v/v) glycerol (Phosphate Buffer, pH 7.0), the homogenate was centrifuged at 240000 xg .min. The floating fat was removed and the microsomal plm supernatant fraction containing 3000 mg protein and 7000 enzyme units in 200 ml Phosphate Buffer (pH 7.0) was decanted and brought to 25% saturation with saturated (NH&O, solution, pH 7.0. After stirring for I h, the suspension was centrifuged at I .IO~ xgmin and the sedimented protein redissolved in IOO ml of Phosphate Buffer (pH 7.0) over a period of 4 h. The turbid solution containing 240 mg protein and 3300 enzyme units was dialysed for 12 h against 2 1 of Phosphate Buffer (pH 7.0) and applied to a column (25 cmxz cm) of DEAE-23 cellulose which had been equilibrated with Phosphate Buffer (pH 7.0). Protein was eluted using a linear gradient (500 ml) of Phosphate Buffer (pH 7.0) in which the concentration of potassium phosphate was increased from 50 to 700 mM. AcetylCoA carboxylase was eluted at 200 mM potassium phosphate. Fractions containing the highest activity acetyl-CoA carboxylase were pooled (zg mg protein and 2600 enzyme units) and the enzyme brought to 25% saturation with saturated (NH&SO, soln., pH 7.0. The precipitated protein was redissolved in 5 ml Phosphate Buffer (pH 7.0) by stirring for 12 h. Undissolved protein was removed by centrifugation at I .106xg.min. The turbid supernatant (II mg protein) contained The enzyme was routinely taken to this 2100 units of acetyl-CoA carboxylase. stage of purification for use in the studies reported here. When stored at o’in Phosphate Buffer (pH 7.0) there was no loss of activity over 2 weeks. No fatty acid synthetase activity could be detected in the enzyme preparation, and it was estimated to contain less than 0.001 units of fatty acid synthetase activity per unit of acetyl-CoA carboxylase activity. Attempts to purify the enzyme further by chromatography on columns of Sagavac 4B or Sepharose 4B which had been equilibrated with Phosphate Buffer (pH 7.0) or with 50 mM Tris-HCl containing 20 mM potassium citrate, I mM dithiothreitol, 0.1mM EDTA and 20% (v/v) glycerol (final pH 7.0), resulted in complete inactivation of the enzyme. Purijication

of lactating-rabbit mammary

gland fatty acid synthetase

The preparation of this enzyme and the criteria to establish its purity have been described3. The enzyme was stored in 0.25 M potassium phosphate buffer (pH 7.0) containing I mM dithiothreitol, I mM EDTA and ZO~/~(v/v) glycerol at -20'. The purified enzyme contained less than 0.03 unit/mg of acetyl-CoA carboxylase activity. Enzyme

assays

Acetyl-CoA carboxylase was assayed by %O, fixation using a method based on that described previously7. Substrate concentration curves were used to determine the apparent Michaelis-Menten constants in the presence of varying amounts of fatty acid synthetase and acetyl-CoA carboxylase (see Table IV) as follows: Preincubation was carried out for 15 min at 37’ in a final volume of 0.1 ml containing 200 mM potassium phosphate buffer (pH 7.0). 50 mM potassium citrate, 6.6 mM MnCI,, 2 mM dithiothreitol, 0.2 mg bovine serum albumin, acetyl-CoA (IOBiochin:. Biophys.

Acta,

248

(1971)sgI-4oj

H. J. M. HANSEN et al.

394

500 PM) and enzyme (up to 4 units). Acetyl-CoA was included in the preincubation medium to simplify the procedure needed to start the reaction. The reaction was

started by the addition of ATP, NaHXO,, (see Table IV) and potassium phosphate final concn.

o-4 units of purified fatty acid synthetase buffer (pH 7.0) in 0.1 ml, so as to give a

(in 0.2 ml) of 200 mM potassium

phosphate

buffer

(pH 7.0),

25 mM

potassium citrate, 3.3 mM MnCl,, I mM dithiothreitol, 0.1 mg bovine serum albumin, 5-250 ,uM acetyl-CoA, IO mM ATP and IO mM NaHlKO, (I ,uC/pmole). Incubations were in duplicate

and incubations

without

acetyl-CoA

were always

included

as

controls. The reaction was stopped with 0.1 ml 5 M perchloric acid, the tubes vigorously agitated for I min and centrifuged. 0.1 ml of 0.1 M NaHCO, was added, the tubes

reagitated

to flush out residual

X0,

and recentrifuged.

procedure was repeated twice. Portions of the final supernatant radioactivity by liquid scintillation counting7. Fatty

acid synthetase

of [r,3-14C,]malonyl-CoA Identification

of reaction

was assayed spectrophotometrically3

using optimum products

This

flushing

were assayed

for

or by incorporation

assay condition9.

other than long-chain

fatty

acids

In experiments where tF< reaction products of [I-lK]acetyl-CoA carboxylation (Table I) or [I,3-14C,]malonyl-CoA utilisation (Table II) were studied, the incubations were stopped with 0.1 ml of 5 M perchloric acid. Samples of reaction products could be stored in 2 M perchloric acid at -20~ for up to 2 months without any detectable change in composition. Protein

was removed by centrifugation

and the supernatant

diluted with water

to give a conductivity identical to that of 0.02 M LiCl in 0.005 M HCl. The sample (approx. 30 ml) was applied to a column of DEAF-23 cellulose (25 cm x I cm) which had been equilibrated with 0.02 M LiCl in 0.005 M HCl. Fractions (approx. were eluted with a linear gradient (500 ml) of LiCl (0.02-0.12 M) in 0.005 at room temperature. A portion (0.5 ml) of each fraction was taken radioactivity7. Fig. I shows a typical elution profile obtained.

IO ml) M

for counting

HCl of

The column was characterised by separately determining the elution profiles of acetic, acetoacetic and malonic acids (free acids, Fraction F), acetyl-CoA, butyrylCoA (C. R. STRONG,

personal

communication)

I

and acetoacetyl-CoA

(acyl-CoA

esters,

I

500

F 400 F ._ E 2 a u

30

200 I

Froctior.

No.

Fig. I. The pattern of reaction products (other than long-chain fatty acids) eluted from a DEAE-ZJ cellulose ion-exchange column after incubation with [r-Wlacetyl-CoA as substrate (see Table I). Biochim.

Biophys.

Ada,

248 (1g71)

391-405

FATTY ACID BIOSYNTHESIS. VIII

395

Fraction A), and malonyl-CoA (Fraction M). In some cases, Fraction F was further characterised by lyophilisation and the proportion of residual non-volatile radioactivity determined. Recovery of the radioactivity applied to the column was routinely 8o-gooj. The incorporation of radioactive substrates into each of the components was determined from the relative proportions of radioactivity in the products. RESULTS

The properties

of acetyl-CoA

carboxylase

From its activity profile on sucrose gradient centrifugation (Fig. z) rabbit mammary gland acetyl-CoA carboxylase appears to be a high mol. wt. protein. Its sedimentation coefficient of approx. 40 S is similar to that of the polymeric form of the enzyme isolated from other tissues*. The presence of a single activity peak on sucrose gradient centrifugation supports earlier evidence0 that the distribution of the enzyme between the microsomal and particle-free supernatant fractions of rabbit mammary gland homogenates is due to its partial sedimentation with the microsomal fraction at 6.2.1o~xg.min. Like chicken liver acetyl-CoA carboxylases the enzyme can be isolated in an active, high mol. wt. form. However, preincubation under conditions which result in activation of the enzyme from chickens and ratlo+” liver, caused only slight changes in activity and no changes in the sedimentation properties of the mammary gland enzyme.

z E$

‘0

i .; sp k ? z Y

5

0

1

2

3 4

5

6

7

8

9

10

11

12 13 14

Fraction No.

15

16

17

Fig. 2. Sucrose gradient centrifugation of partially purified acetyl-CoA carboxylase. hcetyl-CoA carboxylase (43 nmoles/min per 740 ,ug protein) in 0.4 ml Phosphate Buffer (pH 7.0) was layered on a linear sucrose gradient (5-zoo% (w/v), 18 ml) containing Phosphate Buffer (pH 7.0). Centrifugation was carried out at 80000 x g and 20’ for IO h in an MSE 65 centrifuge using a 3 x 20 ml swing-out rotor. Fractions were collected from the bottom and portions were taken for the determination of acetyl-CoA carboxylase activity (see METHODS) and proteina. Purified rabbit mammary gland fatty acid synthetase (s”~~,~ = 16.4 S) and catalase (sO~~,~= II S) were sedimented in separate tubes and the mid-points of their elution profiles are indicated.

The carboxylation

of acetyl-CoA

The carboxylation of acetyl-CoA linked to fatty acid synthesis by the presence of fatty acid synthetase and NADPH (Table Ia) was characterised by an almost total lack of malonyl-CoA accumulation during the incubations. At the two lowest substrate concns. used, the added acetyl-CoA was incorporated into water-soluble free acids (Fraction F, see METHODS) indicating hydrolysis of acetyl-CoA and/or Biochim.

Biophys.

Acta, 248

(1971)

391-405

3

J‘0,

: w ‘Z 2 z

b 6 P

’

&

FATE

UP CAXBOXYLATED

jI-‘dC;hCE’WL-~O~

: I3XjFLCT

OF FATTY

ACID

SYNTHETASE

IN

THE

PRESl?NCE

AND ABSENCE

OF

NADPN

i

+

f

i

-

-

-

-

-

r8o

6

6n

r80

60

6

.;

18,

-^

..~..

Zj6

47

4

18 r&z

(3

36 r54

60

13 <:4

6 18

1;

s- .I

-t-

-+

i

+

i-

+

-+.

+

@

1;1

-” ~--._--~ l_l_l-___ ~ --.-~-~-* Geometrical mean I;: V/initial x tinal concn. ** ~~~er-solub~e CoA derivatives other than makmyf-CoA.

__I~__.

(c)

(b)

(4

__.______~

I.4

0.8

0.3

4.0

3.7

1.2

. ._._.-

0.0

a.0

0.0

2.8

0.4

0.0

2.x

-~

27.4 -.

7.7

0.4

28.8

7.7

0.0

26.2

_(.*

0.0

r.9 2.2

0.0

Q.Q

0.0

0.4 0.6

0.0

-_---

_~~

7.2

3.5

Q,5

o__i

0.2

0.0

0.2

co.2

0.0

0.0

-

3.0 5.x 5.3

0.8

--

Incubation conditions were as described for the assay of acetyl-CoR carboxylase (see METKOT~S) except that unlabelled Nal-ICO, and jr-*4C]acetyKoA (specific act&+, 4.6 p C/F mole) were used and the reaction was stopped with a.2 ml of 5 Y HClU, without subsequent flushings. All incubations were lor IO min. Fatty acid synthetase (a units) and NADPN (40 ,L& were added as shown (+). Four identical incubations in (a) were stopped with or ml of +.I”/&(w/v) NaOH for determination of long-chain fatty acid synthesisr. Patty acids were analysed by gas-Quid radiocbromatography~ and shown to be mainly myristic and palmitic acids. Components other than long-chain fatty acids in incubations stopped with ITCIO, were assayed by DEAEcelhrlose chromatography as described in MI~TNODS. Geometrical means of the substrate concns. are given, since these are appropriate when dealing with the exponential changes encountered in first order kinetics. Where the substrate concentration at the end of the reaction was zero, the geometrical mean is expressed as being < the algebraic mean. __~_~~__l~“-l--l.~----. ~l_--~l. ----.~------~_~.~~_.___._~___ Zjpe Q’ ~nc~4b~t~o~ nmoles acet&CoA recovered as _--.._----~ _---__,__.~~._~~_..___.___“~__~_~~_ AGii$ZX-@atty acid NA DPW A d&d Mean* Cosn$anents with relatzve elufion volumes of: Long-chain I_--~--~-____~____” synthetase acetyk-CoA acetyl- CoA carboxylasc 0.06 o.aB I.00 I.25 fatty acids !Fn-ll (PM) (face acid) ~‘ac+CoA * *) (n%alon?/l-GoA) _” -_~l __I__~.-~-._I-~.~-. _“..-___l ._-. ~~. -., ~~_ -_---

THE

5:

!z IE)

x

i

cr

FATTY ACID BIOSYNTHESIS. VIII

malonyl-CoA,

and into long-chain

,uM, a slight inhibition mulation of in Fraction rather than When

397

of fatty

fatty

acids. At a mean acetyl-CoA

acid synthesis

occurred

concn.

concomitant

of 154

with the accu-

a small amount of malonyl-CoA. The major part of the radioactivity F was found to be non-volatile, suggesting the formation of malonic acetic acid. NADPH was omitted from the incubations so as to prevent the formation

of fatty acids (Table Ib) there was again no substantial malonyl-CoA pool. At the lowest substrate concentration all the radioactivity was found in Fraction F, which showed that fatty acid synthetase As the substrate

concentration

had catalysed increased,

the hydrolysis

the major

of all acyl-CoA esters.

part of Fraction

F was found

to be non-volatile (the same tendency as was observed in Table Ia), indicating the formation of malonic acid. Table Ic shows the results obtained under conditions normally used in the assay of acetyl-CoA carboxylase, i.e. in the absence of fatty acid synthetase and NADPH. Malonyl-CoA was formed at all the substrate concentrations used. However, at the two lowest substrate concentrations, the formation than the incorporation of carboxylated [I-Xlacetyl-CoA corresponding

linked reactions

(cf. Table

Ia),

suggesting

of malonyl-CoA was less into fatty acids in the that

fatty

acid synthetase

had “trapped” the carboxylated [I-%]acetyl-CoA by incorporating it into fatty acids. At a mean acetyl-CoA concentration of 156,uM, more malonyl-CoA was formed (7.2 nmoles) than had been incorporated into fatty acids (5.3 nmoles) in the corresponding linked reaction. Therefore, the inhibition of fatty acid synthesis observed in Table inhibition

Ia in the presence of increasing concentrations of acetyl-CoA is due to of fatty acid synthetase by acetyl-CoA and not to substrate inhibition of

acetyl-CoA carboxylase. In the absence of fatty

acid synthetase

(Table

Ic), the degree

to free acids by the preparation of acetyl-CoA carboxylase lower then when fatty acid synthetase was present (Table of substrate hydrolysis by the acetyl-CoA carboxylase 20% of the rate of carboxylation (Table Ic). The fate of added [r,3-14C,]malonyl-CoA The incubation conditions described

of hydrolysis

alone was found to be I, a and b). The rate

preparation

in Table IIa correspond

alone was approx.

to those normally

used in the assay of fatty acid synthetase with added [r,3-14C,]malonyl-CoA. The highest rate of fatty acid synthesis (0.8 nmole/min) occurred at an acetyl-CoA concentration of about 27 ,uM, and a mean malonyl-CoA concentration of 8 ,uM. In comparison, the highest rate of fatty acid synthesis (0.6 nmole/min) achieved in the linked reaction using [I-14C]acetyl-CoA as substrate (Table Ia) was at a mean acetylCoA concentration of 36 PM. The mean concentration of added [r,3-X,]malonylCoA required to give a rate of synthesis corresponding to 0.6 nmole/min in Table I(a) can be calculated as 3,uM*, which is equivalent to a final malonyl-CoA concentra* The small difference in acetyl-CoA concentrations in the two incubation systems has no effect on the rate of fatty acid biosynthesis and the rate of synthesis is proportional to added malonvlCoA up to 20 pM and enzyme-protein up to 10 units of &tivity3. Therefore, since at 8 pM malo&CoA in Table II the rate of fatty acid synthesis is 0.8 nmoleimin, the concentration of malonvlCoA required to give a rate of 0.6 nmolejmin would be 6 PM. Since in Table I twice the amount‘of fatty acid synthetase activity was used as in Table II, the conwntration of malonyl-CoA required to give a rate of 0.6 nmole/min is 1/2x 6 = 3 ,uM. Biochim.

Biophys.

Acta,

248 (1971) 391-405

II

IS

THE

PRESENCE

OF

FATTY

ACID

SYNTHETASE:

EFFECTS

OF

ADDING

NADPH AND

NON-FUNCTIONING

ACETYL-CoA

.___._

:

-

+

+

+

+ + +

+

+

+

+

+

+

+ 5’

28 100 14 54


IO0 IO

:

28

IO

* Geometrical mean = V//initial; final concn. (see Table I). * * Unknown compounds. * * * Water-soluble CoA derivatives other than malonyl-CoA.

(d)

~

+

-

(c)

5 t$

9 23 94

-

-1

-

(b)

<5 8 58

9 23 94

r;

+ I-

+

+-

_ -

(PM)

(PM)

(4 -

malonyl-CoA

Mean *

Added malonyl-CoA

NA DPH

Acetyl-CoA carboxylase

Fatty acid synthetase

Tyfie of incubation

-

0.7

0.0 0.3 0.4

~___

2.2

I.5 3.8 II.3

0.0 0.7

0.6 0.3 1.3

1.1

I.0

2.9 8.7

0.8 2.4

0.4

I.9

I.5 6.2

0.0

0.5 5.5

0.2

0.7 3.6 10.5

0.7 7.9 0.7 1.3 8.2

0.0

0.0

(malonyl-CoA)

I.2i

of:

0.7 7.2

** *)

volumes

1.00 (acyl-CoA

0.2

0.3

0.4 I.1

0.0 0.5 2.4

0.0 0.2

0.19 +o..& + o.@* *

as

elution

recovered

with relative

0.06 (free acid)

Components

nmoles malonyl-CoA

0.4 I.7 3.3

4.2 3.1

2.1

Long-chain fatty acids

Conditions were as in Table I except that ATP and NaHCO, were omitted from the incubations (so that the acetyl-CoA carboxylase present did not carboxylate substrate) and a solution containing I .O mM acetyl-CoA and 0.85 mM [r,3-14C,]malonyl-CoA (0.9 &/pmole) was used to provide substrates Incubations were for 5 min. Acetyl-CoA carboxylase (0.9 unit), fatty acid synthetase (I unit) and NADPH (40 pg) were added as shown (+) Assays of products were as in Table I. It is assumed that half of the original radioactivity is lost as WO, for all components isolated other than the added [x,3-WZ,]malonyl-CoA. The long-chain fatty acids isolated were not analysed by gas-liquid radiochromatography.

CARBOXYLASE

THE FATE OF [I,3-‘“c,]MALONYL-00.~

TABLE

F w

FATTY ACID BIOSYNTHESIS. VIII

399

tion >6 ,uM, since the initial malonyl-CoA calculated

concentration

value differs very significantly

was zero (see Table I). This

from the final malonyl-CoA

actually found (
concentration

that a substantial

part

of the added [r,3-KJmalonyl-CoA had been converted into components (predominantly Fraction A) other than long-chain fatty acids. The proportion of radioactivity in Fraction A was somewhat increased when NADPH was omitted from the incubations

(Table

IIb),

CoA concns. The incubations preparation The results

reflecting

in Table

the corresponding

increase

II, a and b, were repeated

in the mean malonyl-

with the addition

of the

containing non-functioning acetyl-CoA carboxylase (Table 11,~ and d). of Table II (a-d) are regrouped in Table III according to a factorial

scheme to show the separate effects of non-functioning acetyl-CoA carboxylase and fatty acid synthetase on the formation of acyl-CoA esters isolated as Fraction A. Both fatty acid synthetase and the preparation containing non-functioning acetylCoA carboxylase were able to decarboxylate malonyl-CoA. However, the rate of decarboxylation

by fatty

acid synthetase

alone increased

when the added malonyl-CoA increased responding rate of extra decarboxylation acetyl-CoA the kinetic preparations TABLE

from

1.0 to 7.7

nmoles

from 6.3 to ZI nmoles, whereas the cordue to the addition of non-functioning

carboxylase remained constant at 2.4 and 2.3 nmoles. This indicates that properties for the decarboxylation of malonyl-CoA by the two enzyme were not identical.

III

THE FORMATIONOF ACYL-COA

ESTERS

BY

FATTY

ACID

SYNTHETASE

AND

NON-FUNCTIONING

ACETYL-

CoA CARBOXYLASE The results in Table II have been regrouped in a factorial scheme to show the effects of added malonyl-CoA, NADPH and non-functioning acetyl-CoA carboxylase on the formation of acylCoA esters isolated as Fraction A (see METHODS). The concentrations of added malonyl-CoA in Table II correspond to nmoles added malonyl-CoA in Table III after correction for slight differences in incubation volumes. Added malonyl-CoA (nnzoles) 2.1

Recovered as acyl-CoA esters (nmoles) NA DPH

Acetyl-CoA

Difference

-1.

1.0

-

I.5

Mean 6.3

+ -

Mean 21

carboxylase

+

+

Mean

2.9 3.8 3.7 II.3

0.0

0.7 0.4 0.7 I.3

I.0 0.8

0.9 2.2

2.5

1.0

24

7.2 8.2 7.7

I.5 3.1 2.3

At the mean malonyl-CoA concentrations of 3 and 8 ,uM in Table IIc, there was a marked decrease in the incorporation of malonyl-CoA into fatty acids compared with the corresponding incorporations without added acetyl-CoA carboxylase in Table IIa. This decrease is parallelled in Table III by an increase in the formation of acyl-CoA esters on the addition of partially purified acetyl-CoA carboxylase to incubations initially containing 2.1 and 6.3 nmoles of malonyl-CoA. It would seem Biochim. Biophys. Acta, 248

(1971)391-405

H. J. M. HAiYSEN etd.

4oo

that at the two substrate concentrations in question, the tendency for decarboxylation of malonyl-CoA by acetyl-CoA carboxylase overrules the tendency for malonylCoA to be converted into fatty acids by fatty acid synthetase. Acetyl-CoA carboxylase is thus able to inhibit fatty acid synthesis from added malonyl-CoA by competing for substrate. Apparent

Michaelis-Menten

constants joy acetyl-CoA carboxylase

A marked overall decarboxylation of malonyl-CoA was observed (Table II) under conditions normally used in the assay of acetyl-CoA carboxylase. This indicates that the kinetics of malonyl-CoA formation will involve the concentration of malonylCoA formed in the incubation, which in turn will depend on the amount of enzyme added. Malonyl-CoA formation can be described by the reversible reaction Acetyl-CoA + carboxylase + acetyl-carboxylase + malonyl-CoA + carboxylase

+ malonyl-carboxylase

+ (I)

which is susceptible to product inhibition. Furthermore, an observed decarboxylation by fatty acid synthetase suggests that if any of this enzyme is present in the carboxylase assay it also would give rise to an apparent inhibition of acetyl-CoA carboxylation by the reaction Malonyl-CoA + synthetase + acetyl-CoA + synthetase

+ malonyl-synthetase

--z acetyl-synthetase

+ (2)

The results presented in Fig. 3 demonstrate that addition of fatty acid synthetase leads to an apparent competitive inhibition of the carboxylation of acetylCoA by acetyl-CoA carboxylase. A series of apparent Michaelis-Menten constants (K,) of acetyl-CoA carboxylase for acetyl-CoA at varying amounts of acetyl-CoA carboxylase and added fatty synthetase, and at two different incubation times, are presented in Table IV. A wide range of K, values was obtained, demonstrating that not only fatty acid synthetase, but also acetyl-CoA carboxylase itself gives rise to to incubation competitive inhibition of r4C0, fixation. This inhibition is proportional time. The results in Table IV can be fitted (by graphical extrapolation) into the time in min and i is the equation K, = 12 [I+(~/z.I) .i] where t is the incubation sum of the activity units of acetyl-CoA carboxylase and fatty acid synthetase. The K,, of acetyl-CoA carboxylase from lactating-rabbit mammary gland is, by extrapolation to either zero incubation time or zero protein, 12 PM acetyl-CoA. The apparent Ki of z activity units x min is common for both acetyl-CoA carboxylase and fatty acid synthetase. In terms of malonyl-CoA turnover it is equivalent to z nmole malonyl-CoA formed or utilized in the 0.2 ml incubation volume. Thus Ki can be expressed as an inhibitor concentration, i.e. IO ,uM malonylCoA. Reaction I is described in a simplified manner by the overall rate equationI for the carboxylation of acetyl-CoA. KMV, [acetyl-CoA]

1: = - ~___.

KAKpI+K*

pK~V_,

[malonyl-CoA]+Knl

[malonyl-CoA] racetyl-CoA]

(1)

where V/1and V-, are the maximum velocities from left to right and from right to left, respectively, while KA and KIMare the corresponding Michaelis--Menten constants Biochim.

Biophys.

Ada,

248

(1971) 391-405

FATTY ACID BIOSYKTHESIS.

401

1’111

40 i

32-

‘/[AAc~:>+.CCM] MM-‘! Fig. 3. Double reciprocal plots (LINEWEAVER AND BuRK’~) of acetyl-CoA carboxylase activity uwsus substrate concentration. Incubations were for 5 min with (O-O) and without ( x -x ) the addition of 3.0 units of fatty acid synthetase activity. Enzyme assays were as in METHODS. A-A and c-n are corresponding calculated values from rate equations based on a mechanism of product inhibition (see text).

the absence of competing substrate. From the above we have KA = 12 ,uM acetylCoA and KM = IO ,uM malonyl-CoA. In Fig. 3 the quantitative aspects of this model have been related to experimental results from incubations with acetyl-CoA carboxyl-

in

ase alone.

V, is seen directly

to be equal to 0.35 nmole/min.

V-i can be calculated

from Eq. I by inserting pairs of corresponding substrate concentrations. nmole/min gives the best fit between found and calculated values. Reaction v=

2

is described

= 0.6

by the simplified rate equationi

KAl/, [malonyl-CoA]

KAKM+KA

IL,

[malonyl-CoA]+Ksi

[acetyl-CoA]

(II)

As above KM = IO ,uM malonyl-CoA. V, and KA are both unknown quantities. A combination of Eqn. I and Eqn. II has been used to relate calculated and experimentally found results in Fig. 3 for the incubations with added fatty acid synthetase. V, = 14 nmoles/min and KA = 5 ,uM acetyl-CoA give the best fit between found and calculated values. These results show that the synthetase has a marked capacity for decarboxylation of malonyl-CoA in the absence of acetyl-CoA and NADPH. Biochim. Biofihys. Acta,

248 (1971) 391-405

H. J. M. HANSEX et al.

402 TABLE

IV

APPARENT MICHAELIS-MENTE~

CONSTANTS

FOR

A~ETYL-CoACARBOXYLASE

Enzyme assay conditions were as in Fig. 3, Enzyme activities were varied by altering the amount of enzyme protein added. The amount of acetyl-CoA carboxylase in incubations designated (b) was IO times that in incubations designated (a), the latter being the same as in Pig. 3. When two incubation times were used, the Ir values quoted for acctyl-CoA carboxylase (determined as in Fig. 3) refer to mean values obtained. The activity of the fatty acid synthetase added was determined using optimum assay conditions. ___~_~ Activity units Acetyl-CoA carbo.~ylase (1’)

Fatty acid synthetase (units added)

3.5 (b) 3.6 VJ)

3.1 3.1 4.6 3.0

0.1

0.3 (a) 3.6 (b! 3.5 (b) 1.4

0.1 3.0 3.0

3.0 0.0 0.0 0.3 0.4 0.4

0.2 (4

0.2 0.02 0.2

200

‘6.5 33 30

0.1

0.2

0.3 0.2

200

45

14

140 IO0 IO0 IO0 IO0 IO0 25 20 IX '7 15

DISCUSSION

Our results show that during fatty acid synthesis from acetyl-CoA, a pool of malonyl-CoA does not accumulate. The characteristic properties of the individual enzymes involved in fatty acid synthesis do not favour the net synthesis of malonylCoA. Carboxylated acetyl-CoA is either directly incorporated into fatty acids or catabolized by hydrolysis and decarboxylation. The rapid hydrolysis

of acetyl-CoA

by fatty

acid synthetase

(Table

I) may

be related to the high affinity of fatty acid synthetase for this substrate3. When incorporation of acetate bound to fatty acid synthetase is prevented, acetyl groups are released into solution as acetic acid14. Hydrolysis of malonyl-CoA by fatty acid synthetase occurs under similar conditions14. It is interesting that the hydrolysis of generated malonyl-CoA (Table I) was greater than that of added malonyl-CoA (Table II). This could reflect a difference in mechanism of hydrolysis. Both acetyl-CoA carboxylase and fatty acid synthetase were able to decarboxylate malonyl-CoA (Table II). The conversion of [r,3-14C,]n~alonyl-CoA to other products was always accompanied by a loss in the total amount of radioactivity added. Therefore, any substantial rate of enzyme catalysed transcarboxylation4,15 of 14C0, from [r,3-r*C,]malonyl-CoA to acetyl-CoA could be ruled out. Experiments with highly purified acetyl-CoA carboxylase from chicken liver4+16 and a particle-free supernatant fraction from rat adipose tissue 17showed that the rate of decarboxylation of malonyl-CoA to acetyl-CoA is stimulated by citrate and acetyl-CoA, and could approach 10% of the rate of carboxylation of acetyl-CoA. Using the results in Table III, the maximum rate of malonyl-CoA decarboxylation for rabbit mammary gland Rioclzin~.Biofihys. Acta, ~48 (1971) 391-4Oj

FATTY ACID BIOSYNTHESIS. VIII acetyl-CoA

carboxylase

403

can be calculated

to be approx.

13% of the rate of carboxyla-

tion of acetyl-CoA. Some insight into the nature o< the decarboxylation of malonyl-CoA by fatty acid synthetase is furnished by our previous results2 on the stoichiometry of fatty acid synthesis from acetyl-CoA and from malonyl-CoA. These results showed a preferential utilisation of decarboxylated malonyl-CoA as primer in fatty acid synthesis, indicating a close connection between decarboxylase activity and the incorporation of malonyl-CoA into fatty acids. Malonyl-CoA decarboxylase activity, giving acetyl-CoA as product, co-purified with rabbit mammary gland fatty acid synthetases

and may be an integral

part of the multienzyme

showed that malonyl-CoA is decarboxylated thetase and that the rate of decarboxylation

complex.

LYNEN et ~1.~~

while bound to yeast fatty acid synincreased when fatty acid synthesis

was inhibited. Malonyl-CoA

decarboxylation, catalysed by fatty acid synthetase, occurs also by condensation of malonyl-CoA with “primer” acetyl-CoA during fatty acid synthesis in the presence of NADPH, and during the formation of acetoacetyl-CoA and

triacetic

lactone in the absence of NADPH 3,18m21.The ion-exchange

chromatographic

method we have used did not distinguish between acetyl-CoA, acetoacetyl-CoA, butyryl-CoA and other water-soluble monocarboxylic-CoA esters. Therefore, when fatty acid synthetase was present in incubations, we could not differentiate between decarboxylation of malonyl-CoA to acetyl-CoA and the conversion of malonyl-CoA to acyl-CoA esters other than acetyl-CoA. However, the results in Table III enable us to determine the relative rates of malonyl-CoA decarboxylation by acetyl-CoA carboxylase and fatty acid synthetase at different malonyl-CoA concentrations. The higher rate of decarboxylation by fatty acid synthetase and the different substrate concentration curves obtained can be explained by the additional decarboxylation of malonyl-CoA during the formation of acyl-CoA esters other than acetyl-CoA. Since both acetyl-CoA carboxylase and fatty acid synthetase decarboxylate malonyl-CoA, it would be expected that both enzymes would give rise to an apparent inhibition of malonyl-CoA formation. We have found that this inhibition is, in fact, competitive and that the enzymes seem CREGOLIN et aLs have shown competitive reaction

of acetyl-CoA

to have a similar inhibitory potential. inhibition by malonyl-CoA of the part-

carboxylase

Enzyme-biotin-CO,+acetyl-CoA

+ malonyl-CoA+enzyme-biotin

From their results one can calculate a Kg of about 15 ,uM malonyl-CoA. If this value also applies to the overall carboxylation reaction, it would agree with our calculated value of about IO ,&I malonyl-CoA. The fact that our results concerning the competition between acetyl-CoA and malonyl-CoA do not agree with recent findings of HASHIMOTO AND NUMA~~can be attributed to the high ATP concentration (IO mM) which we have employed. A mechanism of product inhibition will affect the specific activity of acetylCoA carboxylase assayed by malonyl-CoA formation. The true specific activity can be determined independently of any apparent enzyme inhibition by extrapolation of the specific activities measured at different protein concentrations or times of incubation to “zero protein” or “zero time”. The use of initial reaction rates in enzyme

assays

approximates

to this extrapolation,

but is usually

used to ensure a

Biochinz. Biophys. Acta, 248 (1971) 391-405

H. J. hf.HANSEN et a/.

404

constant amount of substrate rather than to avoid conversion of product. DAKSHINAMURTIAND DESJARDINS~’ have pointed out that the assay of acetyl-CoA carboxylase in the cytosol of rat adipose tissue requires measurement of the initial reaction rate to ensure that it is unaffected

by any decarboxylase

activity.

SCORPIO AND MASOROZ”

have reported the influence of malonyl-CoA decarboxylation on the assay of acetylCoA carboxylase in rat liver cytosol. When the carboxylation of acetyl-CoA is linked to fatty acid synthesis by the addition of NADPH and fatty acid synthetase (Table Ia), the most striking observation is that fatty acid synthetase no longer acts as an inhibitor of this carboxylation. Fatty acids were synthesised at a rate equivalent to that of acetyl-CoA carboxylation, suggesting a mechanism which specifically promotes the incorporation of carboxylated acetyl-CoA

into fatty

acids and prevents

its catabolism

by side reactions.

At the

same time there was no pool of free malonyl-CoA to account for the rate of fatty acid synthesis observed. This would agree with a metabolic compartmentation such as the direct formation of malonyl-S-enzyme by the carboxylation of acetyl-Senzyme2s2*. In the presence of NADPH, malonyl-S-enzyme is converted into fatty acids; otherwise it is gradually decomposed by decarboxylation and hydrolysis. A mechanism of metabolic compartmentation could also consist in the formation of malonyl-CoA close to fatty acid synthetase. The apparent localisation of substrate in the vicinity of glycolytic enzymes has been discussed elsewhere25. Early data of WAKIL et aLz6 with partially purified enzymes of avian liver show that there is no initial time-lag for the spectrophotometric assay of fatty acid synthesis from acetylCoA. This would support our finding that there is a lack of intermediate malonylCoA accumulation in the linked reaction. The concentration of malonyl-CoA has been considered to be a parameter controlling the chain-length of fatty acids synthesised in dyo27-2Y. In view of the results

in this paper,

it would be more appropriate

to consider

the rate of acetyl-

CoA carboxylation as the regulatory factor in chain-length control. A search of the literature has failed to reveal any data on the concentration of malonyl-CoA

in tissues.

ACKNOWLEDGE&lESTS We are indebted to the late Professor G. Hiibscher for his encouragement and interest. We thank Dr. P. K. Tubbs, University of Cambridge, for useful discussions, colleagues in the Department for advice and Miss Beryl Dixon for excellent technical assistance. The Medical Research Council of Great Britain provided financial support. H. J.M.H. was on leave of absence from the Danish Atomic Energy Commission.

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2x0 (1970) loo

4 C. GREGOLIN, E. RYDER AND M. D. LANE, J. Biol. Chem., ~43 (1968) 4227. 5 A. L. MILLER AND H. R. LEVY, J. Bid. Chem., 244 (1969) 2334. 6 D. J. EASTER AND R. DILS, Biochim. Biophys. Acta, 152 (1968) 653. 7 S. SMITH, D. J, EASTER AXD R. DILS, Biochim. Biophys. Ado, 125 (1966) 445. 8 A. K. KLEINSCHMIDT, J, Moss AND M. D. LANE, Science,166(1969) 1276. R&him.

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FATTY

ACID

BIOSYNTHESIS.

VIII

4%

A. K. KLEIXSCHMIDT AND M. LANE, Proc. Nat!. 9 C. GREGOLIN, E. RYDER, R. C. WARNER, Acad. Sci. U.S., 56 (1966) 1751. S. N~MA AND E. RINGELMAN,B~OC~~UZ. Z., 343 (1965) 258. IO W. P. WELBOURNE, R. F. SWAXSON AND H. S. ANKER, Biochem. Biophys. Res. Common., 37 II (1969) 933. AND D. BURK, J. Am. Chem. Sot., 56 (1934) 658. I2 H. LINEWEAVER ‘3 J. M. REINER,~~M. FLORKIN AND E. G. STOTZ, Comprehensive Biochemistry, Vol. 12, Elsevier, New York, 1964, p. 126. ANI) J. W. PORTER,J. Biol. Chem., z39(1964) 1346. 1-i J. D. BRO~IE,G.WASSON F.LYNEN, Biochem.Z.,340(1964) 22X. 15 S. NUMA,E.RINGELMANAND 16 E. RYDER, C. GREGOLIN, H. CHAXG APZD M. D. LAXE, Proc. Natl. Acad. Sci. U.S., 57 (1967) ‘455. K. DAKSHINAMURTIAKD P.R.DESJARDINS, Biochim.Bioprhys.Acta, 176(1969) 221. F.LYNEX, I.HOPPER-KESSEL AND H. EGGERER, Biochem.Z., 340(1964) 95. D.B. MARTIN,M.G.HORNINGANDP.R.VAGELOS,J.B~~~. Chem., 236(1961)663. R. BRESSLER AND S. J. WAKIL, J. Biol. Chem., 236 (1961) 1643. C. J. CHESTERTON, I?.H. W. BUTTERWORTH APED J. W. PORTER, Arch. Biochem. Biophys., 126 (1968) 864. 18(1971) 319. 22 T. HASHIMOTO AND S. NUMA, Euv.J.Biochem., E.J.MAsoRo, Bi0chem.J.. 118(1970)391. 23 R.M.SCORPIOAND AND I,.G.HANSEN, ActaChem..Scand.,23(1969) 2180. 24 H.J.M.HAR.sEN Bioenrrgetics, 2 (1971) 115. 25 G. H~~BSCHER, R. J. MAYER AND H. J. M. HANSEN, .I. 26 S. J. WAKIL, E. B. TITCHENER AND D. M. GIBSON, Biochim.Biophys. Ada, 34 (1959)227. Acta, 116 (1966) 23. 27 S. SMITH AND R. DILS, Biochim.Biophys. AND I. L. CHAIKOFF, Biochim.Biophys. Acta, 144 (1967) 51. 28 J. C. BARTLEY, S. ABRAHAM ANDF. LYNEN, Eur..f.Biochem., 10(1g69) 377. 29 M. SUMPER,D.OESTERHELT,C.RIEPERTINGER Biochim. Biophys. Acta, 248 (1971) 391-405