THE JounsAL OF BIoLoGICaL CHEMISTRY

Vol 247, X‘o. 2, Issue of January 25, pp. GOJ-~10, 1972

Printed in U.S.A.

Communications The incubation mixture contained

phate buffer, pH 7.0 (50); 2-mercaptoethanol
(in micromoles) : potassium
(25); NADPH
phos-
(5); mxlo-
nyl-CoA (2); [lW]acetyl-CoA (1.0, 18,000, or 29,000 cps) ; [l-l%]-
butyryl-CoA (1.0, 26,600 cps) ; and enzyme, 5 mg, 0.0 to 0.4 ammonium
sulfate precipitate, in a total volume of 1.0 ml. Incubation time was
15 min at 37”. The fatty acids formed were extracted, chromato-
graphed, and counted as described earlier (2).
Pathway for the Synthesis of Fatty Acids
Primer incorporation
in Mammalian Tissues Enzyme source
(Received for publication, October 18, 1971) Acetyl-CoA Butyryl-CoA
! -y
CHU YUAN LIN* AND SOMA KUMAR amoles/mg pro1ein/min
Rat mammary gland.. . . . . .. . 0.60 2.10
From the DepartwLent of Chemistry, Georgetown University,
Rat liver. .. . . . . .. . . . 0.35 1.26
Washington, D. C. 20007 Bovine mammary gland. . .. .. 0.97 1.57
Rabbit liver. . . . . . 0.25 0.93
Rat adipose tissue. .... .. . . 1.27 0.43
SUMMARY Pigeon liver.. . .. . . . . . 3.8 0.07

Fatty acid synthetase of the liver of all of the mammalian

species investigated and of the lactating mammary gland TABLE II
shows a pronounced preference for butyryl-CoA over acetyl- Distribution of primer X’ in fat@ acids
CoA as primer in the synthesis of fatty acids from the primer, The incubation mixture contained (in micromoles) : potassium phos-
phate buffer, pH 7.0 (50) ; 2.mercaptoethanol (25); NADPH (5) ; NADH
malonyl-CoA and NADPH. This relative primer specificity,
(5); malonyl-CoA (3.1); [l-W]acetyl-CoA (1.6, 30,000 cps) or [l-iaC]-
in contrast to that of the enzyme complex isolated from rat butyryi-CoA (2.0, 53,200 cps); and 1.0 ml of particle-free supernatant.
adipose or pigeon liver, appears to be related to the presence Total volume was 1.75 ml. Incubation period was 45 min. The fatty
acids formed were extracted and chromatographed, and an aliquot of
in the particle-free supernatant of enzymes which are capable the acids longer than butyrate was subjected to Kuhn-Roth oxidation
of synthesizing butyryl-CoA from acetyl-CoA by the reversal after adding 10 mg of palmitic acid as carrier, as described in the text.
of p oxidation. A high proportion of fatty acid synthesis
aetyl-GA
from acetyl-CoA by the mammalian liver and mammary onverted to
butyryl-
supernatant, which contained NADH, NADPH and malonyl- Fatty Acetate CoA prior
Electron acids re-
CoA, occurred after its initial conversion to butyryl-CoA. donor Primer to con-
xidized cwerec densation
with
malonyl-
The presence of ,Eketoacyl-CoA thiolase, NADH-dependent /3- CoA
ketoacyl-Cob reductase, and enoyl-CoA hydratase in the cytosol
CPS %
of lactating rabbit mammary tissue, besides their occurrence in Rabbit mam- NADPH+ [1-W]Acetyl- 700 385 90
the mitochondria, was demonstrated in an earlier report (1). mary gland NADH CoA
It was further shown that these enzymes, in the presence of fatty
Rabbit mam- NADPH+ [l-W]Acetyl- 365 168 92
acid synthetase, which had an enoyl-CoA reductase activity mary gland NADH CoA
associated with it, synthesized butyryl-CoA from acetyl-CoA by
Rabbit mam- NADPH [1-W]Acetyl- 795 729 16.6
the reversal of p oxidation (1, 2). For the sake of convenience,
mary gland CoA
we propose to call these reactions /3 reduction. Rabbit mammary
fatty acid synthetase was also observed to have a pronounced Rat liver NADPH+ [l-i4C]Acetyl- 225 125 89
NADH CoA
preference for butyryl-CoA over acetyl-CoA as primer and to
synthesize 2 to 3 times more fatty acids from acetyl-CoA in the Pigeon liver NADPH’ [l-i4C]Acetyl- 984 912 14.6
presence of the three soluble ,f-oxidative enzymes (2). It ap- NADH CoA

peared from these observations that acetyl-CoA was first con- Pigeon liver NADPH [1-i4C]Acetyl- 1179 1107 12.2
verted to butyryl-CoA by 0 reduction and its carbon chain CoA
elongated further by the NADPH-dependent sequential addition
Rat adipose NADPH+ [l-i4C]Acetyl- 436 404 14.5
of the carbon atoms from malonyl-CoA catalyzed by the fatty NADH CoA
acid synthetase. The relative primer specificity exhibited by
Rat adipose NADPH [1-W]Acetyl- 556 512 15.8
the rabbit (2) and goat (3) mammary synthetase is also shown CoA
by the rat mammary (4) and potato (5) enzymes. We have
extended the work to determine whether this primer specificity Rabbit mam- NADPH+ [1-‘*C]Butyryl- 5560 84
mary gland NADH CoA
is a characteristic of the enzyme from mammary tissue, among - -
animal tissues, and also to determine whether the P-reductive
reactions have a role in the synthesis of fatty acids in tissues of various tissues was ascertained from the rate of incorporation
other than mammary gland. of the two primers using the enzyme fraction obtained by its
The relative primer specificity of the fatty acid synthetase precipitation at 40y0 saturation of ammonium sulfate from the
respective particle-free supernatant. The procedure used for
* The results presented in this communication are to be included in a
doctoral dissertation to be submitted to Georgetown University. the preparation of the supernatant and the substrates, determi-

604

This is an Open Access article under the CC BY license.

Issue of January 25, 1972 C. Y. Lin and X. Kumar 605

nation of protein concentration, isolation of fatty acids formed, Roth oxidation of the fatty acids was corrected for the loss of
and assay of the enzyme were as described earlier (2). The data 10% of this acid. The synthesis of fatty acids from acetyl-CoA
presented in Table I show that the enzyme from liver and mam- with butyryl-CoA as an intermediate would be expected to re-
mary gland of each of the mammalian species investigated shows sult in acids with 14C label equally distributed between carbon
a preference for butyryl-CoA. In contrast, the enzyme from rat atoms 2 and 4 counted from the methyl end of the chain. These
epididymal fat pad exhibited the reverse primer specificity. acids would be mixed with acids formed from acetyl-CoA used
Pigeon liver enzyme, as reported earlier by Wakil (6), utilized directly as primer, which would contain the 14C label in only the
butyryl-CoA to a relatively insignificant extent only. second carbon from the methyl end. Thus, if the loss in radio-
Investigation of the acetyl-CoA-dependent NADH oxidation activity on Kuhn-Roth oxidation is multiplied by 2, the frac-
(I) in the dialyzed supernatants of different tissues revealed tion of the species with label on both carbons would be obtained,
that at least the thiolase and P-ketoacy-CoA reductase are pres- which would represent the fraction of the fatty acids formed
ent in lactating mammary gland, as well as the liver of each of from acetyl-Coii after its conversion to butyryl-CoA.
the mammalian species, but are absent in adipose tissue. It Data presented in Table II show that in rabbit mammary and
appears, therefore, that the preference for butyryl-CoA as primer rat liver supernatants containing both TU’ADH and NADPH,
is related to the presence of the @-oxidative enzymes in the 89 to 92% of acetyl-CoA was converted to butyryl-Co-1 by 6
supernatant. reduct.ion before further chain elongation by condensation with
For the determination of the extent of conversion of acetyl- malonyl-CoA. When the P-reductive steps were blocked by
CoA to butyryl-CoA by the P-reductive reactions before chain the omission of NADH from the incubation mixture containing
elongation by the synthetase, the fraction of fatty acids, con- rabbit mammary supernatant, the fraction of fatty acids calcu-
taining 6 or more carbon atoms synthesized from [l-14C]acetyl- lated to contain 14C in carbons 2 and 4 decreased to 16.6%.
CoA by the particle-free supernatant, was subjected to Kuhn- Values similar to the latter were obtained with pigeon liver and
Roth oxidation by the procedure of Ginger (7) with certain rat adipose supernatants, both in the presence and a,bsence of
modifications. These consisted in oxidizing the acids, contain- NADH, confirming the a.bsence of the P-oxidative enzymes in
ing carrier palmitic acid, with chromic acid in a sealed vial at these tissues. The apparent presence of 12 to 16% of fatty
120” for 4 hours. The mixture was cooled in ice, and excess acids with label in 2 carbon atoms, where /3-oxidative enzymes
chromic acid was reduced with hydrazine hydrate and diluted are either absent or inoperative, cannot be accounted for at
with 25 ml of water; 8 g of magnesium sulfate was then added and present. It cannot arise from incomplete oxidation of the acids,
distilled until mass crystallization of the salt. The distillate containingthelabelin carbon 4 (counting from the methyl end),
(approximately 23 ml) was neutralized, concentrated, and forming volatile acids other than acetic, as the oxidation of the
assayed for radioactivity. The collection and evaporation of a fatty acids formed from [1-14C]butyryl-CoA resulted in the re-
large volume of steam distillate were thus avoided. Recovery covery of very little labeled acetic acid (Table II). This loss
of [l-14C]acetate, subjected to the entire procedure, was con- of 14C on oxidation of acids, where none should have occurred,
sistently 90 to 92y0. Hence, acetic acid obtained after Kuhn- might represent the extent of loss of the 2 methyl end carbon

,B-ketoacyl-CoA
2 Acetyl-S-CoA Acetoacetyl-S-CoA + CoASH
thiola&

a-ketoacyl-CoA \
Acetoacetyl-S-CoA + NADH + H+ T
reductase

P-hydroxybutyryl-S-CoA + NAD+

enoyl-CoA ,
$-hydroxybutyryl-S-CoA 1 Crotonyl-S-CoA + H20
hydratase

fatty acid synthetase ,or

Crotonyl-S-CoA + NADPH + H+ \
enoyl-CoA reductase

Butyryl-S-CoA + NADP+

fatty acid
a
Butyryl-S-CoA + nMalonyl-S-CoA
- + 2nNADPH
- + 2nHf
- synthetase

CH3CH2CH2CH2-(CH2-CH2)n_l-CH2COOH + ZfiNADP+ +n+lCoASH

SCHEME 1
 Synthesis of Fatty Acids Vol. 247, No. 2

atoms of acids during oxidation or the formation of acids by the bearing a structural resemblance to chemically activated inter-
reversal of /3 oxidation (or both), reported to be catalyzed by the mediates in catalysis or incorporating the binding characteris-
fatty acid synthetase itself (8). tics of several reactants (in a multisubstrate reaction) in a single
The possibility that the enzymes causing @ reduction found in inhibitor molecule. We wish to report an inhibitor of carboxy-
the cytoplasm are of mitochondrial origin can be ruled out as peptidase A, apparently of the latter type, which is considerably
neither leakage nor release of the P-oxidative enzymes occurred more tightly bound than any reversible inhibitor of this enzyme
when rabbit, mammary mitochondria were either subjected to previously described.
repeated homogenization or were disrupted by freezing and The present inhibitor, 2(R)-benzyl-3-carboxypropionic acid
thawing (1). The mitochondrial enzymes stayed bound to the (I), was prepared by a procedure similar to that described by
mitochondrial membranes and even when disrupted were re- Cohen and Milovanovic (2). Racemic 2-benzyl-3-carboxy-
moved on ultracentrifugation. Similarly, Williamson et al. (9) propionic acid was first converted to the dimethyl ester (b.p.
have established that acetoacetyl-CoA thiolase, found in rat 145-153” per 3.5 mm; literature 148-151” per 3 mm (3)). Stereo-
liver cytoplasm, did not arise as a result of mitochondrial dis- selective hydrolysis with chymotrypsin gave 2(n)-benzyl-3-
ruption as there was no leakage of glutamate dehydrogenase carbomethoxypropionic acid, which yielded I (m.p. 160-161”,
during the homogenization procedure. It is concluded from the [o(] $ = +26”) upon alkaline hydrolysis. 2(S)-Benzyl-3-car-
results that in the mammalian liver and mammary tissue the boxypropionic acid (m.p. 161-162”, [al]? = -26”) was obtained
important primer in the fatty acid synthetase-catalyzed reactions by alkaline hydrolysis of the chymotrypsin-resistant portion of
is butyryl-CoR and not acetyl-CoA and that the major pathway the racemic dimethyl ester. The properties of both enantiomers
for the synthesis of fatty acids in these tissues involves the reac- were identical with those reported by Cohen and Nilovanovic
tions shown in Scheme 1. To what extent these reactions ac- (2). Other inhibitors were obtained as shown in the footnotes
tually occur under physiological conditions remains to be es- to Table I.
tablished. The activity of carboxypeptidase A from beef pancreas (the
crystalline Anson preparation, obtained from Sigma) was de-
REFERENCES
termined spectrophotometrically with the substrates hippuryl-
1. XANDEDKAR, A. K. N., AND KUMAR, S. (1969) Arch. Biochem. Bio-
phys., 134, 563.
L-phenylalanine and hippuryl-L-phenyllactate at 254 nm (7)
2. LIN, C. T., AND KUMAR, S. (1971) J. Biol. Chem., 246, 3284. and with the substrate carbobenzyloxyglycylglycyl-L-phenylala-
3. NANDEDKAR, A. K. N., SCHIRMER, E. W., PYNADATH, T. I., AND nine at 228 nm where this substrate gave Ae = -330 for hy-
KUMAR, S. (1969) Arch. B&hem. Biophys., 134, 554.
4. SMITH, S., AND ABRAHAM, S. (1971) J. Biol. Chem., 246, 2537.
5. HUANG, K. I’., AND STUMPF, P. K. (1971) Arch. B&hem. Biophys.,
TABLE I
143, 412.
6. WAKIL, S. J. (1961) J. Lipid. Res., 2, 1. Inhibitors of Carborypeptidase Aa
7. GINGER, L. G. (1944) J. Biol. Chem., 156, 453.
8. CAREY, E. M., AND DILS, R. (1970) Biochim. Biophus. Acta, 210, 388. Inhibitor Ki
9. !J~ILI.IAMSON, D. H., BATES, M. W., AND KREBS, H. A. (1968) Bio-
them. J., 108, 353.
L-Benzylsuccinic acid (I) 6 X ;“,- h
n-Benzylsuccinic acid 5 x 10-e
on-Benzylsuccinic acid 1.1 x 10-S
A Potent Reversible Inhibitor of 2(R)-Benzyl-3.carbomethoxypropionic acid (half- G x 10-G h
ester of I)
Carboxypeptidase A* n-2-Methyl-3.phenylpropionic acidb 1.4 x lo-3h
3.l’henylpropionic acid (II)” 1.0 x 10-&h
(Received for publication, October 18, 1971) on-2-Benzylglutaric acidd 5 x 10-G
Benzylmalonic acid” 6 X 1O-5 i
L. 1). BYERS AND R. WOLFERTDEN un-Phenylsuccinic acide 2 x 10-4 j
Succinic acid” 4 x 10-4 i
From the Department of Biochemistry, University of North L-Phenylalanine” 5 x 10”
Carolina, Chapel Hill, North Carolina 27514 Hippuric scidf 8 X lO+h
Carbobenxyloxyglycine 3 x 10-Z h. f
Carbobenzyloxyglycylglycine~ >10-2 h. i
SUMMARY Hippuryl+phenylalaninee (K,) 2 x 10-S
Carbobenzyloxyglycyl-n-phenylalanineg (Ii,) 2 x 10-z
Benzylsuccinic acid and closely related compounds are Carbobenzyloxyglycylglycyl-n-phenylalanineg 8 x 10-d
effective inhibitors of carboxypeptidase A. The “L” isomer M?n)
Hippuryl-L-phenyllactic acid (lcm) 2 x 10-d
of benzylsuccinic acid, Z(R)-benzyl-3carboxypropionic acid,
appears to be bound by the enzyme several orders of magni- a Determined from double reciprocal plots using hippuryl-L-phenyl-
tude more tightly than known substrates and inhibitors. alnnine as substrate in Tris-HCl buffer (0.025 M, pH 7.5, containing
sufficient NaCl to maintain ionic strength 0.5) at 25’, unless otherwise
This inhibitor may resemble the collected substrates for the noted.
reverse reaction, combining their individual binding charac- b Prepared by the action of methyl iodide on diethylbenzylmalonnte
teristics in a single molecule. in sodium ethoxide, followed by hydrolysis, decarboxylation, and resolu-
tion by the method of Schrecker (4).
c Obtained from Eastman Co. and recrystallized.
Reversible inhibitors are useful for probing the binding prop- d Prepared by the method of Ansell and Hey (5).
e Obtained from Aldrich Co. and recrystallized.
erties of enzymes and may also help in elucidating mechanisms f Obtained from Sigma.
of catalysis (1). Unusual potency is expected of inhibitors 0 Obtained from Cycle Co.
h Also tested using carbobenzyloxyglycylglycyl-L-phenylalanine as
* This work was supported in part by Research Grants GM-12725 substrate, with similar results.
and GY-18325, a predoctoral fellowship (G&1-49094), and a research i Ki estimated from the concentration of inhibitor required to produce
career development award (Ahf-08560) from the National Institutes of 50% inhibition.
Health, United States Public Health Service. j Value also obtained by Auld and Vallee (6).