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Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for over-all synthetase activity
ARCHIVES
OF BIOCHEMISTRY
Substrate Optimum
AND
BIOPHYSICS
Inhibition
163, 324-331 (1974)
of Pigeon
Assay Conditions SARVAGYA
Liver Fatty Acid Synthetase for Over-all
S. KATIYAR
AND
JOHN
Synthetase
and
Activity’
W. PORTER
Lipid Metabolism Laboratory, Veterans Administration Hospital and the Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Received December
21, 1974
The effects of the substrates acetyl-CoA, malonyl-CoA, and NADPH on the activity of pigeon liver fatty acid synthetase have been studied over a wide range of concentrations. Double-reciprocal coordinate plots for each of the substrates have been found to be linear at low concentrations. At higher concentrations two of the substrates, acetyl-CoA and malonyl-CoA, inhibit the rate of fatty acid synthesis. This double substrate inhibition is apparently of a competitive type. Inhibition by acetyl-CoA is very strong as compared to that by malonyl-CoA. At a 4: 1 ratio of acetyl- to malonyl-CoA, inhibition is about 75%, whereas at a 4: 1 ratio of malonyl- to acetyl-CoA fatty acid synthesis proceeds at the maximum rate. These results are consistent with the hypothesis that a competition between acetyl-CoA and malonyl-CoA occurs for the occupany of the 4’. phosphopantetheine site, a prosthetic group of the synthetase complex, and possibly also for the hydroxyl binding site (or sites), The relative concentrations of these substrates and the binding constants for each then determine whether these sites are occupied by acetyl or malonyl groups, and whether inhibition of fatty acid synthesis occurs. Based on our results, assays for pigeon liver fatty acid synthetase activity should be conducted at substrate concentrations of 15 PM, 60 FM, and 100 pM for acetyl-CoA, malonyl-CoA, and NADPH, respectively.
The pigeon liver fatty acid synthetase multienzyme complex catalyzes the synthesis of long-chain fatty acids from acetyland malonyl-CoA in the presence of NADPH (1). This enzyme system has three sites that convalently bind acetyl groups (from acetyl-CoA) and two that bind malonyl groups. These have been identified as a hydroxyl site (possibly serine), 4’-phosphopantetheine and possibly cysteine (2-4). In view of the common sites (hydroxyl and 4’-phosphopantetheine) for the binding of acetyl and malonyl groups, one would expect that acetyland malonyl-CoA would compete with each other for the occupancy of these common sites. However, the mutual inhibitory effects of ‘This investigation was supported in part by Research Grant, AM 01383, from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service. 324
these substrates on the formation of fatty acids have not yet been studied in detail. Plate et al. (5), while studying the role of fructose 1,6-diphosphate in the stimulation of fatty acid synthesis, reported that double-reciprocal plots of l/v against l/ [malonyl-CoA] were nonlinear at subinhibitory levels of malonyl-CoA and that the rate of synthesis of fatty acids reduced We have, to zero at 75 PM malonyl-CoA. however, obtained results contrary to these findings. This discrepancy in results is presumably due to the dissociation of fatty acid synthetase complex under the experimental conditions employed by Plate and co-workers. The process of dissociation and reassociation of the fatty acid synthetase complex has recently been studied (6-8) in detail in our laboratory, with the resultant establishment of the conditions under which no dissociation of the complex OCcurs.
SUBSTRATE
INHIBITION
OF FATTY
Lynen and coworkers (9, 10) have studied the inhibition of the fatty acid synthetase multienzyme complex of yeast by long-chain acyl coenzyme A compounds. They have found (9) that CIZ-, C14-, C16-, and C,s-saturated acyl-CoA esters act as competitive inhibitors with respect to malonyl-CoA and noncompetitive inhibitors with respect to acetyl-CoA during the synthesis of fatty acids. However, substrate inhibition by malonyl-CoA has not been observed (10) with the yeast enzyme when acetyl-CoA is the initiator of fatty acid synthesis. This has been interpreted to mean that there is no competition between acetyl-CoA and malonyl-CoA for the same transacylase site. With pigeon liver fatty acid synthetase, however, a different initiation mechanism may be operative since malonyl-CoA and acetyl-CoA are shown in the present study to be mutually inhibitory. The results of our investigation on the effects of substrate concentration on the over-all rate of fatty acid synthesis by the purified pigeon liver fatty acid synthetase complex are reported in this paper. This investigation has resulted in the establishment of the optimum conditions for the assay of activity of this enzyme. EXPERIMENTAL
PROCEDURE
Materials. Acetyl-CoA and malonyl-CoA were obtained from P-L Biochemicals, and NADPH was purchased from Sigma. DTT’ (Cleland’s reagent) was a product of Calbiochem. Other chemicals were obtained as follows: EDTA from Fisher, KH,PO, from Mallinckrodt, and K,HPO, from Matheson, Coleman, and Bell. All other inorganic reagents were of analytical grade, and deionized glass-distilled water was used for all experiments. Apparatus. All spectrophotometric assays were carried out with a Gilford spectrophotometer, Model 24OOS, which was equipped with a thermostated l-cm cell compartment, and with a Model 2475A thermostated 5cm cuvette compartment. All light-absorption spectra were recorded with a Beckman DKBA ratio recording spectrophotometer. A radiometer pH meter, Model 51, was used for pH measurements of buffers and other solutions.
Methods Enzyme synthetase
purification. was purified
*The following DTT.
The pigeon liver fatty acid by the procedure of Hsu,
abbreviation
is used: dithiothreitol,
ACID SYNTHESIS
325
Wasson, and Porter (1) as modified by Butterworth et al. (6). However, 2-mercaptoethanol was replaced by 1 mM DTT in all purification steps to eliminate the possibility of oxidation of SH groups of the enzyme (8). Purified enzyme was then stored frozen at -- 20°C in 0.2 M phosphate, 10 mM DTT, and 1 mM EDTA in an atmosphere of nitrogen. Enzyme protein was determined by the biuret method of Gornall, Bardawill, and David (11). of fatty acid synthetase at full Maintenunce activity during the experiment. A stock solution of the fatty acid synthetase of the desired concentration was made after the frozen enzyme was thawed and then incubated at 25°C for about 1 hr. The stock solution, 0.2 M in phosphate and 10 mM in DTI’, was stored at 25°C throughout the experiment to maintain full enzyme activity. Periodic assay of the activity of the stock solution of enzyme showed that no decrease in activity, and thus no dissociation of the enzyme, occurred during the experiment. Assay of fatty acid synthetase actiuit,y. Fatty acid synthetase activity was measured by following the decrease in light absorbance of NADPH at 340 nm. In the spectrophotometric assay in l-cm cells, the reaction mixture contained 100 pM NADPH, 1 mM DTT, 3 mM EDTA, 0.2 M phosphate (potassium) buffer, pH 7.0, varying amounts of acetyl-CoA and malonyl-CoA (specified in the legends to figures and in the text), varying amounts of fatty acid synthetase complex, 2-8 fig of protein, in a total volume of 1 .O ml. In assays with the 5-cm cell, the total volume of the reaction was kept to 5.0 ml and the concentrations of all components were the same as for the l-cm cell, except that NADPH was either 30 or 40 pM and enzyme protein was 2 Fg per ml. The reaction was started by the addition of enzyme to the mixture of substrates equilibrated at 25°C for 5 min. The initial slope of the recorder tracing was utilized to calculate the rate of fatty acid synthesis. Determination of substrate concentrations prior to Assam. The concentrations of acetyl-CoA and malonyl-CoA were calculated from their absorbance at 260 nm (c = 15.4 x 10’ Mm’ cm-‘) whereas the concentration of NADPH was calculated from its absorbance at 340 nm. All substrate solutions were prepared fresh prior to the start of each experiment and they were stored in ice during the progress of the experiment. The absence of free CoA in malonyl-CoA and acetyl-CoA preparations was shown by assays by the method of Ellman (12). RESULTS
Effect of NADPH concentration on the rate of synthesis of fatty acids. The rate of
fatty acid synthesis increased with increasing concentration of NADPH as shown in Fig. 1A. The increase in the rate is high at lower concentrations of NADPH and it
326
KATIYAR
AND PORTER
,A /’ 0
20
IO NADPH
30
(FM)
0’ 0
I 01
I 02
I 03
-0 135 NAOFH (,,M)-’
FIG. 1. Effect of NADPH concentration on the rate of fatty acid synthesis in 0.2 M phosphate buffer at pH 7.0. Each reaction mixture contained potassium phosphate buffer, pH 7.0, 0.2 M; DTT, 1 mM; EDTA, 3 mM; acetyl-CoA, 30 PM; malonyl-CoA, 50 PM; purified fatty acid synthetase, 2 fig protein per ml; and varying concentrations of NADPH and water. The total volume of the reaction mixture was 5 ml. Mixtures without enzyme were incubated for 5 min at 25°C and the reaction was then started by the addition of enzyme separately thermostated at 25’C. The rate of the reaction was followed spectrophotometrically in a 5-cm cell as described under Experimental Procedure. (A) Velocity units on the ordinate are nmoles of NADPH oxidized per min per mg protein.
decreases as NADPH concentration is progressively increased. At 20 PM NADPH the rate of fatty acid synthesis is about 85% of the maximum value and thereafter even large increases in NADPH concentration result in relatively small increases in the rate of the reaction (data at NADPH concentrations higher than 30 ELM are not shown in the figure). At concentrations of NADPH higher than 100 PM, no further effect on the rate of fatty acid synthesis was observed. The same pattern of saturation of the rate with increasing concentration of NADPH was observed when the levels of acetyl-CoA and malonyl-CoA were varied between 1 and 30 ELM and 1 and 100 PM, respectively. The double-reciprocal plot (Fig. 1B) of the data of Fig. 1 indicates that the Michaelis-Menten equation is applicable in a narrow range of low concentrations of NADPH where the spectrophotometric assay with a l-cm cell is of little value. For observing the correct changes in rate at concentrations of NADPH less than 15 PM, the use of a 5-cm light path is essential.
tion of the rate of fatty acid synthesis as a function of malonyl-CoA concentration is shown in Fig. 2A. The concentration of acetyl-CoA was maintained at a low level (4 PM) because of its strong inhibitory effect (vide infra) and NADPH was 30 PM in order to make the rates satisfactory for study with a 5-cm path length cell. It can be seen from Fig. 2A that there is a steep rise in the rate up to 8 PM of malonyl-CoA. The rate becomes maximum at 16 pM and then decreases with a further increase in malonyl-CoA concentration. Under these experimental conditions, the maximum synthetase activity was observed when the ratio of acetyl-CoA to malonyl-CoA was 1 to 4. At higher concentrations of malonyl-CoA inhibition occurred. When the concentration of malonyl-CoA was 100 PM; i.e., 25 times higher than that of acetyl-CoA, a 30% inhibition of fatty acid synthesis was observed. This is at variance with the data obtained by Plate et al. (5) who found maximum synthetase activity at an acetyl-CoA to malonyl-CoA ratio of about 3 : 1, and a total inhibition of syntheEffect of malonyl-CoA concentration on tase activity when the concentration of the rate of synthesis of fatty acids. Varia- malonyl-CoA was two times higher than
SUBSTRATE
INHIBITION
OF FATTY
that of acetyl-CoA. On the reciprocal coordinates, linearity was observed only at low concentrations of malonyl-CoA (Fig. 2B). The apparent value of K, for malonyl-CoA is 8.7 PM. These findings are different from bhose of Plate et al. (5) who reported a nonlinear reciprocal plot for all concentrations of malonyl-CoA. Effect of acetyl-CoA concentration on the rate of synthesis of fatty acids. When the rate of fatty acid synthesis was studied as a function of acetyl-CoA concentration, the results shown in Fig. 3A were obtained. At a fixed concentration of malonyl-CoA of 10 PM, the increase in rate of fatty acid synthesis was very high, up to 2 pM of acetyl-CoA. Then the increase became progressively less until a concentration of 5 PM acetyl-CoA was reached, where maximum enzyme activity was found. As the acetyl-CoA concentration was increased further, a rapid decrease in rate of fatty acid synthesis was observed, indicating a very strong substrate inhibition by acetyl-CoA. At acetyl-CoA to malonyl-CoA ratios of 1: 1, 2: 1, 3: 1, and 4: 1 inhibition by acetyl-CoA is 33,40, 70, and 7596, respectively. Inhibition by acetyl-CoA is thus much more marked than that exhibited by malonyl-CoA. A Lineweaver-Burk plot (Fig. 3B) yielded a value of 1 FM for the apparent K,,, of acetyl-CoA. Optimum substrate concentrations for
327
ACID SYNTHESIS
the assay of fatty acid synthetase actiuity. The tolerance of the highest concentration of one substrate at a fixed level of the other and vice versa by fatty acid synthetase prior to the observable inhibitory effects, was studied over a wide range of acetylCoA and malonyl-CoA concentrations. Figure 4A-C presents the results obtained when acetyl-CoA was kept at a fixed level in the range of l-33 PM and malonyl-CoA was varied from l-10 times for each acetylCoA concentration. It is of interest to note that the maximum synthetase activity was found when the ratio of acetyl-CoA concentration to malonyl-CoA was 1: 4. Invariably, in all the assays it can be seen that as malonyl-CoA was increased further to five times and higher than that of acetylCoA, significant inhibition of the rate of fatty acid synthesis was observed. However, when the effect of varying acetyl-CoA concentration on the various fixed concentrations of malonyl-CoA was investigated, very strong substrate inhibition by acetyl-CoA was observed, The results obtained are summarized in Fig. 5A and B. It can be seen from these data that the highest rate of fatty acid synthesis was observed at acetyl-CoA concentrations of 2 and 10 PM, respectively, when malonyl-CoA was held constant at 4 and 20 PM. Thus, it can be concluded that, in general, at a fixed concentration of malonyl-CoA,
120 r A I
0
25
10
20
30
40
50
MALONYL-CoA
60 (,,Ml
-AI 70
LLI 80
90
100
01
02
03
MALONYL-CoA
04 (,,M)-’
FIG. 2. Effect of malony-CoA concentration on the rate of synthesis of fatty acids in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1 except that the concentrations of substrates were as follows: acetyl-CoA, 4 PM, and NADPH, 30 hc~. A 5-cm cell was used for the light absorbance measurements. (A) Velocity units on the ordinate are nmoles NADPH oxidized per min per mg protein.
05
328
KATIYAR
I
0
I
10
I
I
I
20 ACETYL-COA
1
AND PORTER
I
30 (NM)
I
40
I
, 02
I 04
I 06
ACETYL-CoA
I OS
I 10
(JIM)-’
FIG. 3. Rate of fatty acid synthesis as a function of acetyl-CoA concentration at fixed levels of NADPH and malonyl-CoA in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1 except that the concentrations of the substrates were as follows: malonyl-CoA, 10 HIM and NADPH, 30 pM. Rates were followed spectrophotometritally with a 5-cm cell as described in the Experimental Procedure. (A) Velocity units on the ordinate are nmoles NADPH oxidized per min per mg protein.
fatty acid synthetase shows maximum activity when the nonsaturating concentration of acetyl-CoA is one-half of the malonyl-CoA concentration. However, the highest activity for every fixed concentration of acetyl-CoA (including the concentration of acetyl-CoA exhibiting maximum activity at a fixed concentration of malonyl-CoA) is observed when malonyl-CoA is 4fold the concentration of acetyl-CoA. Whenever the ratio of acetyl-CoA to malonyl-CoA is raised to greater than 1: 2 the inhibitory effect of acetyl-CoA becomes very marked.
dent when the ratio of these two substrates was varied. When malonyl-CoA : acetylCoA was 4: 1, the rate was maximum, but at the 4: 1 ratio of acetyl-CoA: malonylCoA, 75% inhibition of the maximum rate was observed. This is further demonstrated by the data of Figs. 2 and 3 where the inhibition increases rapidly with increasing concentration of acetyl-CoA. A slower increase in inhibition is observed when the malonyl-CoA concentration is increased at fixed concentrations of acetylCoA. In view of the observed inhibitory effects DISCUSSION of acetyl- and malonyl-CoA we have optiFatty acid synthetase from pigeon liver mized the concentrations of these subhas been found in the present study to be strates for the assay of over-all activity of sensitive to inhibition by malonyl-CoA and fatty acid synthesis. Figure 6 summarizes acetyl-CoA. The inhibition of fatty acid the results obtained for the assay of differsynthesis by increasing concentrations of ent concentrations of enzyme at various acetyl-CoA at fixed concentrations of mal- 1:4 ratios of acetyl- to malonyl-CoA. It onyl-CoA, and vice versa, is a general may be noted from this figure that the phenomenon and it occurs over a wide maximum rate for the synthesis of fatty range of concentrations of acetyl-CoA and acids is observed at acetyl- and malomalonyl-CoA. The inhibition shown by nyl-CoA concentrations of 15 and 60 PM, malonyl-CoA was much smaller than that respectively. Further increase in the conexhibited by acetyl-CoA. The relative de- centration of these substrates (in the opgree of inhibition of fatty acid synthesis by timum ratio of 1:4) does not make a acetyl- and malonyl-CoA was made evi- significant change. It is, therefore, recom-
SUBSTRATE
0
20
IO MALONYL-
t 0
10
I 20
\ 30
INHIBITION
OF FATTY
30
COP, (,JM)
, 40
I
,
1
/
50
60
70
80
MALONYL-CoA
hb.4)
40
0
‘lo
120 80 MALONYL-CoA
160 (JIM)
200
FIG. 4. Rate of fatty acid synthesis as a function of malonyl-CoA concentration at various levels of acetyl-CoA and a fixed level of NADPH in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1, except that the concentrations of acetyl-CoA were as follows: (A) 1 ELM (A-A), 2 PM (OA), 3 PM (A-A), and 4 NM (O---Cl); (B) 5 pM (O--O), 6 pM (A-A), 8 jtM (@A), and 10 pM (A-A). The concentration of NADPH in (A) and (B) was 30 @M in each assay and assays were carried out in 5-cm cells. Enzyme protein was 2 pg per ml for each assay.
ACID SYNTHESIS
329
mended that for the assay of over-all activity of fatty acid synthetase, acetyl- and malonyl-CoA concentrations be held at 15 and 60 pM, respectively. In an earlier report from this laboratory Chesterton, Butterworth, and Porter (13) concluded from peptic hydrolysis and electrophoresis that the cysteine and 4’-phosphopantetheine sites of the fatty acid synthetase have a high affinity for the acetyl moiety, and that the hydroxyl site shows a preference for the binding of the malonyl group. Subsequent studies by Phillips et al. (4) have led to the proposal that acetyl groups are transferred from acetyl-CoA to the hydroxyl site, then to 4’-phosphopantetheine, and finally to the cysteine site of the enzyme. The malonyl group is transported to the 4’-phosphopantetheine site of the enzyme via the hydroxyl site prior to the condensation reaction (4). Thus, in the reactions that lead to fatty acid synthesis, the 4’-phosphopantetheine, and possibly the hydroxyl sites are utilized by both substrates. It is to be expected, therefore, that a competition between the t,wo substrates would occur. The data of this paper show that this is indeed the result obtained. This behavior of the pigeon liver enzyme is, however, quite different from the yeast enzyme where a similar substrate inhibition by malonyl-CoA in the presence of acetyl-CoA has not been observed (10). The binding constant of acetyl-CoA at the 4’-phosphopantetheine site is about five times higher than the binding constant for malonyl-CoA (13). A higher concentration of malonyl-CoA in comparison to acetyl-CoA would, therefore, be needed to offset the preferential binding of the acetyl moiety at the 4’-phosphopantetheine site. Since the first two steps in the synthesis of fatty acids involve transacylation of acetyl and malonyl moieties from their CoA esters (C) The concentration of acetyl-CoA was 15 WM (O-O), 20 fiM (A--A), and 33 FM (A-A). Enzyme protein was 8 fig and NADPH was 100 PM in each assay. The final volume of the assay mixture was 1 ml. A l-cm cell was used to follow the rate of reaction spectrophotometrically. Units on the ordinate are nmoles of NADPH oxidized per min per mg protein.
330
KATIYAR AND PORTER
I20 i 0
. SO
;
. :I 40
t 0
I 2
1 4
1 6
ACETYL-CoA
I e
I
I
0
IO
(J&V
I IO
I 20
I 30
I 40
ACETYL-CoAQM)
FIG. 5. Fatty acid synthesis as a function of acetyl-CoA concentration at different levels of malonyl-CoA and a fixed level of NADPH in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1, except that the concentrations of malonyl-CoA were as follows: (A) 4 1~ (O---O), 6 WM(A-A), and 8 go (A-A); (B) 10 jtM (O-O), 20 pM (A-A), 30 pM (A-A), and 40 pM (O---O). A 5-cm cell was used for assay. Units on the ordinate are nmoles NADPH oxidized per min per mg protein.
100
80 Y 0
60
; 40
20
0
2 FATTY
4
6
ACID SYNTHETASE
e IN IJ~
FIG. 6. Fatty acid synthetase activity at an acetylCoA: malonyl-CoA ratio of 1:4 at different levels of acetyl-CoA and malonyl-CoA in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 4C. Concentrations of substrates were as follows: acetyl-CoA, 4 PM, malonyl-CoA, 16 pM (04); acetyl-CoA, 5 PM, malonyl-CoA, 20 KM (a---@); acetyl-CoA, 10 pM, malonyl-CoA, 40 pM (O--O); acetyl-CoA, 15 PM, malonyl-CoA, 60 PM (A-A-A); acetyl-CoA, 20 pM, malonyl-CoA, 80 pM (A-A-A); and acetyl-CoA, 30 PM, malonybCoA, 100 &M (O--O--O). The NADPH concentration was 100 PM in each assay. A l-cm cell was used for the assays as described in the Experimental Procedure. Velocity units on the ordinate are nmoles NADPH oxidized per min.
to a hydroxyl site and then to a 4’-phosphopantetheine site, a blockage of either site by the acetyl moiety would result in the inhibition of fatty acid synthesis. Our observation that at concentrations of acetyl-CoA higher than twice the concentration of malonyl-CoA a high inhibition occurs, is therefore consistent with the earlier binding studies (3, 13). The milder inhibition of fatty acid synthesis by malonyl-CoA at concentrations higher than four times the acetyl-CoA concentration observed in the present study is consistent with the fact that the enzyme is capable of synthesizing fatty acids from only malonyl-CoA and NADPH (14). In this process the malonyl moiety is decarboxylated (presumably when bound to 4’-phosphopantetheine) to yield an acetyl moiety. It is evident from our results that the availability of the binding sites or the proper moiety (acetyl or malonyl groups) is of prime importance for the maximum rate of synthesis of fatty acids. The partial blockage of the 4’-phosphopantetheine and possibly the hydroxyl site by an acetyl or a malonyl group results in the competitive inhibition observed by us. Inhibition of the synthetase activity by malonyl-CoA has also been noted by Plate et al. (5). However, the complete inhibition of the synthe-
SUBSTRATE
INHIBITION
OF FATTY
sis of fatty acids at 30 pM acetyl-CoA and reported by these 75 pM malonyl-CoA workers, was not observed by us. It appears in retrospect that Plate et al. (5) were probably working with a partially dissociated synthetase complex since their studies were carried out in the presence of low ionic-strength buffers. It has been shown by us that pigeon liver fatty acid synthetase is completely dissociated into half-molecular weight subunits in low ionic strength Tris-glycine buffer (8).
5.
6.
7.
8. 9.
REFERENCES 1. Hsu, R. Y., WASSON, G., ANDPORTER, J. W. (1965). J. Biol. Chem. 240, 3736-3746. 2. PHILLIPS, G. T., NIXON, J. E., ABRAMOVITZ, A. S., AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 357-371. 3. NIXON, J. E., PHILLIPS, G. T., ABRAMOVITZ, A. S., AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 372-379. 4. PHILLIPS, G. T., NIXON, J. E., DORSEY, J. A., BUTTERWORTH, P. H. W., CHESTERTON, C. J.,
10. 11. 12. 13.
14.
ACID SYNTHESIS
331
AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 380-391. PLATE, C. A., JOSHI, V. C., SEDGWICK, B., AND WAKIL, S. J. (1968) J. Biol. Chem. 243, 5439-5445. BUITERWORTH, P. H. W., YANG, P. C., BOCK, R. M., AND PORTER, J. W. (1967) J. Biol. Chem. 242, 3508-3516. KUMAR, S., DORSEY, J. K., AND PORTER, J. W. (1970) Biochem. Biophys. Res. Commun. 40, 825-832. KUMAR, S., MUESING, R. A., AND PORTER, J. W. (1972) J. Biol. Chem. 247, 4749-4762. LUST, G., AND LYNEN, F. (1968) Eur. J. Biochem. 7, 68-72. AYLING, J., PIRSON, R., AND LYNEN, F. (1972) Biochemistry 4, 526-532. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. CHESTERTON, C. J., BUTTERWORTH, P. H. W., AND PORTER, J. W. (1968) Arch. Biochem. Biophys. 1.26, 864-872. KATIYAR, S. S., BRIEDIS, A. V., AND PORTER, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420.
OF BIOCHEMISTRY
Substrate Optimum
AND
BIOPHYSICS
Inhibition
163, 324-331 (1974)
of Pigeon
Assay Conditions SARVAGYA
Liver Fatty Acid Synthetase for Over-all
S. KATIYAR
AND
JOHN
Synthetase
and
Activity’
W. PORTER
Lipid Metabolism Laboratory, Veterans Administration Hospital and the Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Received December
21, 1974
The effects of the substrates acetyl-CoA, malonyl-CoA, and NADPH on the activity of pigeon liver fatty acid synthetase have been studied over a wide range of concentrations. Double-reciprocal coordinate plots for each of the substrates have been found to be linear at low concentrations. At higher concentrations two of the substrates, acetyl-CoA and malonyl-CoA, inhibit the rate of fatty acid synthesis. This double substrate inhibition is apparently of a competitive type. Inhibition by acetyl-CoA is very strong as compared to that by malonyl-CoA. At a 4: 1 ratio of acetyl- to malonyl-CoA, inhibition is about 75%, whereas at a 4: 1 ratio of malonyl- to acetyl-CoA fatty acid synthesis proceeds at the maximum rate. These results are consistent with the hypothesis that a competition between acetyl-CoA and malonyl-CoA occurs for the occupany of the 4’. phosphopantetheine site, a prosthetic group of the synthetase complex, and possibly also for the hydroxyl binding site (or sites), The relative concentrations of these substrates and the binding constants for each then determine whether these sites are occupied by acetyl or malonyl groups, and whether inhibition of fatty acid synthesis occurs. Based on our results, assays for pigeon liver fatty acid synthetase activity should be conducted at substrate concentrations of 15 PM, 60 FM, and 100 pM for acetyl-CoA, malonyl-CoA, and NADPH, respectively.
The pigeon liver fatty acid synthetase multienzyme complex catalyzes the synthesis of long-chain fatty acids from acetyland malonyl-CoA in the presence of NADPH (1). This enzyme system has three sites that convalently bind acetyl groups (from acetyl-CoA) and two that bind malonyl groups. These have been identified as a hydroxyl site (possibly serine), 4’-phosphopantetheine and possibly cysteine (2-4). In view of the common sites (hydroxyl and 4’-phosphopantetheine) for the binding of acetyl and malonyl groups, one would expect that acetyland malonyl-CoA would compete with each other for the occupancy of these common sites. However, the mutual inhibitory effects of ‘This investigation was supported in part by Research Grant, AM 01383, from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service. 324
these substrates on the formation of fatty acids have not yet been studied in detail. Plate et al. (5), while studying the role of fructose 1,6-diphosphate in the stimulation of fatty acid synthesis, reported that double-reciprocal plots of l/v against l/ [malonyl-CoA] were nonlinear at subinhibitory levels of malonyl-CoA and that the rate of synthesis of fatty acids reduced We have, to zero at 75 PM malonyl-CoA. however, obtained results contrary to these findings. This discrepancy in results is presumably due to the dissociation of fatty acid synthetase complex under the experimental conditions employed by Plate and co-workers. The process of dissociation and reassociation of the fatty acid synthetase complex has recently been studied (6-8) in detail in our laboratory, with the resultant establishment of the conditions under which no dissociation of the complex OCcurs.
SUBSTRATE
INHIBITION
OF FATTY
Lynen and coworkers (9, 10) have studied the inhibition of the fatty acid synthetase multienzyme complex of yeast by long-chain acyl coenzyme A compounds. They have found (9) that CIZ-, C14-, C16-, and C,s-saturated acyl-CoA esters act as competitive inhibitors with respect to malonyl-CoA and noncompetitive inhibitors with respect to acetyl-CoA during the synthesis of fatty acids. However, substrate inhibition by malonyl-CoA has not been observed (10) with the yeast enzyme when acetyl-CoA is the initiator of fatty acid synthesis. This has been interpreted to mean that there is no competition between acetyl-CoA and malonyl-CoA for the same transacylase site. With pigeon liver fatty acid synthetase, however, a different initiation mechanism may be operative since malonyl-CoA and acetyl-CoA are shown in the present study to be mutually inhibitory. The results of our investigation on the effects of substrate concentration on the over-all rate of fatty acid synthesis by the purified pigeon liver fatty acid synthetase complex are reported in this paper. This investigation has resulted in the establishment of the optimum conditions for the assay of activity of this enzyme. EXPERIMENTAL
PROCEDURE
Materials. Acetyl-CoA and malonyl-CoA were obtained from P-L Biochemicals, and NADPH was purchased from Sigma. DTT’ (Cleland’s reagent) was a product of Calbiochem. Other chemicals were obtained as follows: EDTA from Fisher, KH,PO, from Mallinckrodt, and K,HPO, from Matheson, Coleman, and Bell. All other inorganic reagents were of analytical grade, and deionized glass-distilled water was used for all experiments. Apparatus. All spectrophotometric assays were carried out with a Gilford spectrophotometer, Model 24OOS, which was equipped with a thermostated l-cm cell compartment, and with a Model 2475A thermostated 5cm cuvette compartment. All light-absorption spectra were recorded with a Beckman DKBA ratio recording spectrophotometer. A radiometer pH meter, Model 51, was used for pH measurements of buffers and other solutions.
Methods Enzyme synthetase
purification. was purified
*The following DTT.
The pigeon liver fatty acid by the procedure of Hsu,
abbreviation
is used: dithiothreitol,
ACID SYNTHESIS
325
Wasson, and Porter (1) as modified by Butterworth et al. (6). However, 2-mercaptoethanol was replaced by 1 mM DTT in all purification steps to eliminate the possibility of oxidation of SH groups of the enzyme (8). Purified enzyme was then stored frozen at -- 20°C in 0.2 M phosphate, 10 mM DTT, and 1 mM EDTA in an atmosphere of nitrogen. Enzyme protein was determined by the biuret method of Gornall, Bardawill, and David (11). of fatty acid synthetase at full Maintenunce activity during the experiment. A stock solution of the fatty acid synthetase of the desired concentration was made after the frozen enzyme was thawed and then incubated at 25°C for about 1 hr. The stock solution, 0.2 M in phosphate and 10 mM in DTI’, was stored at 25°C throughout the experiment to maintain full enzyme activity. Periodic assay of the activity of the stock solution of enzyme showed that no decrease in activity, and thus no dissociation of the enzyme, occurred during the experiment. Assay of fatty acid synthetase actiuit,y. Fatty acid synthetase activity was measured by following the decrease in light absorbance of NADPH at 340 nm. In the spectrophotometric assay in l-cm cells, the reaction mixture contained 100 pM NADPH, 1 mM DTT, 3 mM EDTA, 0.2 M phosphate (potassium) buffer, pH 7.0, varying amounts of acetyl-CoA and malonyl-CoA (specified in the legends to figures and in the text), varying amounts of fatty acid synthetase complex, 2-8 fig of protein, in a total volume of 1 .O ml. In assays with the 5-cm cell, the total volume of the reaction was kept to 5.0 ml and the concentrations of all components were the same as for the l-cm cell, except that NADPH was either 30 or 40 pM and enzyme protein was 2 Fg per ml. The reaction was started by the addition of enzyme to the mixture of substrates equilibrated at 25°C for 5 min. The initial slope of the recorder tracing was utilized to calculate the rate of fatty acid synthesis. Determination of substrate concentrations prior to Assam. The concentrations of acetyl-CoA and malonyl-CoA were calculated from their absorbance at 260 nm (c = 15.4 x 10’ Mm’ cm-‘) whereas the concentration of NADPH was calculated from its absorbance at 340 nm. All substrate solutions were prepared fresh prior to the start of each experiment and they were stored in ice during the progress of the experiment. The absence of free CoA in malonyl-CoA and acetyl-CoA preparations was shown by assays by the method of Ellman (12). RESULTS
Effect of NADPH concentration on the rate of synthesis of fatty acids. The rate of
fatty acid synthesis increased with increasing concentration of NADPH as shown in Fig. 1A. The increase in the rate is high at lower concentrations of NADPH and it
326
KATIYAR
AND PORTER
,A /’ 0
20
IO NADPH
30
(FM)
0’ 0
I 01
I 02
I 03
-0 135 NAOFH (,,M)-’
FIG. 1. Effect of NADPH concentration on the rate of fatty acid synthesis in 0.2 M phosphate buffer at pH 7.0. Each reaction mixture contained potassium phosphate buffer, pH 7.0, 0.2 M; DTT, 1 mM; EDTA, 3 mM; acetyl-CoA, 30 PM; malonyl-CoA, 50 PM; purified fatty acid synthetase, 2 fig protein per ml; and varying concentrations of NADPH and water. The total volume of the reaction mixture was 5 ml. Mixtures without enzyme were incubated for 5 min at 25°C and the reaction was then started by the addition of enzyme separately thermostated at 25’C. The rate of the reaction was followed spectrophotometrically in a 5-cm cell as described under Experimental Procedure. (A) Velocity units on the ordinate are nmoles of NADPH oxidized per min per mg protein.
decreases as NADPH concentration is progressively increased. At 20 PM NADPH the rate of fatty acid synthesis is about 85% of the maximum value and thereafter even large increases in NADPH concentration result in relatively small increases in the rate of the reaction (data at NADPH concentrations higher than 30 ELM are not shown in the figure). At concentrations of NADPH higher than 100 PM, no further effect on the rate of fatty acid synthesis was observed. The same pattern of saturation of the rate with increasing concentration of NADPH was observed when the levels of acetyl-CoA and malonyl-CoA were varied between 1 and 30 ELM and 1 and 100 PM, respectively. The double-reciprocal plot (Fig. 1B) of the data of Fig. 1 indicates that the Michaelis-Menten equation is applicable in a narrow range of low concentrations of NADPH where the spectrophotometric assay with a l-cm cell is of little value. For observing the correct changes in rate at concentrations of NADPH less than 15 PM, the use of a 5-cm light path is essential.
tion of the rate of fatty acid synthesis as a function of malonyl-CoA concentration is shown in Fig. 2A. The concentration of acetyl-CoA was maintained at a low level (4 PM) because of its strong inhibitory effect (vide infra) and NADPH was 30 PM in order to make the rates satisfactory for study with a 5-cm path length cell. It can be seen from Fig. 2A that there is a steep rise in the rate up to 8 PM of malonyl-CoA. The rate becomes maximum at 16 pM and then decreases with a further increase in malonyl-CoA concentration. Under these experimental conditions, the maximum synthetase activity was observed when the ratio of acetyl-CoA to malonyl-CoA was 1 to 4. At higher concentrations of malonyl-CoA inhibition occurred. When the concentration of malonyl-CoA was 100 PM; i.e., 25 times higher than that of acetyl-CoA, a 30% inhibition of fatty acid synthesis was observed. This is at variance with the data obtained by Plate et al. (5) who found maximum synthetase activity at an acetyl-CoA to malonyl-CoA ratio of about 3 : 1, and a total inhibition of syntheEffect of malonyl-CoA concentration on tase activity when the concentration of the rate of synthesis of fatty acids. Varia- malonyl-CoA was two times higher than
SUBSTRATE
INHIBITION
OF FATTY
that of acetyl-CoA. On the reciprocal coordinates, linearity was observed only at low concentrations of malonyl-CoA (Fig. 2B). The apparent value of K, for malonyl-CoA is 8.7 PM. These findings are different from bhose of Plate et al. (5) who reported a nonlinear reciprocal plot for all concentrations of malonyl-CoA. Effect of acetyl-CoA concentration on the rate of synthesis of fatty acids. When the rate of fatty acid synthesis was studied as a function of acetyl-CoA concentration, the results shown in Fig. 3A were obtained. At a fixed concentration of malonyl-CoA of 10 PM, the increase in rate of fatty acid synthesis was very high, up to 2 pM of acetyl-CoA. Then the increase became progressively less until a concentration of 5 PM acetyl-CoA was reached, where maximum enzyme activity was found. As the acetyl-CoA concentration was increased further, a rapid decrease in rate of fatty acid synthesis was observed, indicating a very strong substrate inhibition by acetyl-CoA. At acetyl-CoA to malonyl-CoA ratios of 1: 1, 2: 1, 3: 1, and 4: 1 inhibition by acetyl-CoA is 33,40, 70, and 7596, respectively. Inhibition by acetyl-CoA is thus much more marked than that exhibited by malonyl-CoA. A Lineweaver-Burk plot (Fig. 3B) yielded a value of 1 FM for the apparent K,,, of acetyl-CoA. Optimum substrate concentrations for
327
ACID SYNTHESIS
the assay of fatty acid synthetase actiuity. The tolerance of the highest concentration of one substrate at a fixed level of the other and vice versa by fatty acid synthetase prior to the observable inhibitory effects, was studied over a wide range of acetylCoA and malonyl-CoA concentrations. Figure 4A-C presents the results obtained when acetyl-CoA was kept at a fixed level in the range of l-33 PM and malonyl-CoA was varied from l-10 times for each acetylCoA concentration. It is of interest to note that the maximum synthetase activity was found when the ratio of acetyl-CoA concentration to malonyl-CoA was 1: 4. Invariably, in all the assays it can be seen that as malonyl-CoA was increased further to five times and higher than that of acetylCoA, significant inhibition of the rate of fatty acid synthesis was observed. However, when the effect of varying acetyl-CoA concentration on the various fixed concentrations of malonyl-CoA was investigated, very strong substrate inhibition by acetyl-CoA was observed, The results obtained are summarized in Fig. 5A and B. It can be seen from these data that the highest rate of fatty acid synthesis was observed at acetyl-CoA concentrations of 2 and 10 PM, respectively, when malonyl-CoA was held constant at 4 and 20 PM. Thus, it can be concluded that, in general, at a fixed concentration of malonyl-CoA,
120 r A I
0
25
10
20
30
40
50
MALONYL-CoA
60 (,,Ml
-AI 70
LLI 80
90
100
01
02
03
MALONYL-CoA
04 (,,M)-’
FIG. 2. Effect of malony-CoA concentration on the rate of synthesis of fatty acids in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1 except that the concentrations of substrates were as follows: acetyl-CoA, 4 PM, and NADPH, 30 hc~. A 5-cm cell was used for the light absorbance measurements. (A) Velocity units on the ordinate are nmoles NADPH oxidized per min per mg protein.
05
328
KATIYAR
I
0
I
10
I
I
I
20 ACETYL-COA
1
AND PORTER
I
30 (NM)
I
40
I
, 02
I 04
I 06
ACETYL-CoA
I OS
I 10
(JIM)-’
FIG. 3. Rate of fatty acid synthesis as a function of acetyl-CoA concentration at fixed levels of NADPH and malonyl-CoA in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1 except that the concentrations of the substrates were as follows: malonyl-CoA, 10 HIM and NADPH, 30 pM. Rates were followed spectrophotometritally with a 5-cm cell as described in the Experimental Procedure. (A) Velocity units on the ordinate are nmoles NADPH oxidized per min per mg protein.
fatty acid synthetase shows maximum activity when the nonsaturating concentration of acetyl-CoA is one-half of the malonyl-CoA concentration. However, the highest activity for every fixed concentration of acetyl-CoA (including the concentration of acetyl-CoA exhibiting maximum activity at a fixed concentration of malonyl-CoA) is observed when malonyl-CoA is 4fold the concentration of acetyl-CoA. Whenever the ratio of acetyl-CoA to malonyl-CoA is raised to greater than 1: 2 the inhibitory effect of acetyl-CoA becomes very marked.
dent when the ratio of these two substrates was varied. When malonyl-CoA : acetylCoA was 4: 1, the rate was maximum, but at the 4: 1 ratio of acetyl-CoA: malonylCoA, 75% inhibition of the maximum rate was observed. This is further demonstrated by the data of Figs. 2 and 3 where the inhibition increases rapidly with increasing concentration of acetyl-CoA. A slower increase in inhibition is observed when the malonyl-CoA concentration is increased at fixed concentrations of acetylCoA. In view of the observed inhibitory effects DISCUSSION of acetyl- and malonyl-CoA we have optiFatty acid synthetase from pigeon liver mized the concentrations of these subhas been found in the present study to be strates for the assay of over-all activity of sensitive to inhibition by malonyl-CoA and fatty acid synthesis. Figure 6 summarizes acetyl-CoA. The inhibition of fatty acid the results obtained for the assay of differsynthesis by increasing concentrations of ent concentrations of enzyme at various acetyl-CoA at fixed concentrations of mal- 1:4 ratios of acetyl- to malonyl-CoA. It onyl-CoA, and vice versa, is a general may be noted from this figure that the phenomenon and it occurs over a wide maximum rate for the synthesis of fatty range of concentrations of acetyl-CoA and acids is observed at acetyl- and malomalonyl-CoA. The inhibition shown by nyl-CoA concentrations of 15 and 60 PM, malonyl-CoA was much smaller than that respectively. Further increase in the conexhibited by acetyl-CoA. The relative de- centration of these substrates (in the opgree of inhibition of fatty acid synthesis by timum ratio of 1:4) does not make a acetyl- and malonyl-CoA was made evi- significant change. It is, therefore, recom-
SUBSTRATE
0
20
IO MALONYL-
t 0
10
I 20
\ 30
INHIBITION
OF FATTY
30
COP, (,JM)
, 40
I
,
1
/
50
60
70
80
MALONYL-CoA
hb.4)
40
0
‘lo
120 80 MALONYL-CoA
160 (JIM)
200
FIG. 4. Rate of fatty acid synthesis as a function of malonyl-CoA concentration at various levels of acetyl-CoA and a fixed level of NADPH in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1, except that the concentrations of acetyl-CoA were as follows: (A) 1 ELM (A-A), 2 PM (OA), 3 PM (A-A), and 4 NM (O---Cl); (B) 5 pM (O--O), 6 pM (A-A), 8 jtM (@A), and 10 pM (A-A). The concentration of NADPH in (A) and (B) was 30 @M in each assay and assays were carried out in 5-cm cells. Enzyme protein was 2 pg per ml for each assay.
ACID SYNTHESIS
329
mended that for the assay of over-all activity of fatty acid synthetase, acetyl- and malonyl-CoA concentrations be held at 15 and 60 pM, respectively. In an earlier report from this laboratory Chesterton, Butterworth, and Porter (13) concluded from peptic hydrolysis and electrophoresis that the cysteine and 4’-phosphopantetheine sites of the fatty acid synthetase have a high affinity for the acetyl moiety, and that the hydroxyl site shows a preference for the binding of the malonyl group. Subsequent studies by Phillips et al. (4) have led to the proposal that acetyl groups are transferred from acetyl-CoA to the hydroxyl site, then to 4’-phosphopantetheine, and finally to the cysteine site of the enzyme. The malonyl group is transported to the 4’-phosphopantetheine site of the enzyme via the hydroxyl site prior to the condensation reaction (4). Thus, in the reactions that lead to fatty acid synthesis, the 4’-phosphopantetheine, and possibly the hydroxyl sites are utilized by both substrates. It is to be expected, therefore, that a competition between the t,wo substrates would occur. The data of this paper show that this is indeed the result obtained. This behavior of the pigeon liver enzyme is, however, quite different from the yeast enzyme where a similar substrate inhibition by malonyl-CoA in the presence of acetyl-CoA has not been observed (10). The binding constant of acetyl-CoA at the 4’-phosphopantetheine site is about five times higher than the binding constant for malonyl-CoA (13). A higher concentration of malonyl-CoA in comparison to acetyl-CoA would, therefore, be needed to offset the preferential binding of the acetyl moiety at the 4’-phosphopantetheine site. Since the first two steps in the synthesis of fatty acids involve transacylation of acetyl and malonyl moieties from their CoA esters (C) The concentration of acetyl-CoA was 15 WM (O-O), 20 fiM (A--A), and 33 FM (A-A). Enzyme protein was 8 fig and NADPH was 100 PM in each assay. The final volume of the assay mixture was 1 ml. A l-cm cell was used to follow the rate of reaction spectrophotometrically. Units on the ordinate are nmoles of NADPH oxidized per min per mg protein.
330
KATIYAR AND PORTER
I20 i 0
. SO
;
. :I 40
t 0
I 2
1 4
1 6
ACETYL-CoA
I e
I
I
0
IO
(J&V
I IO
I 20
I 30
I 40
ACETYL-CoAQM)
FIG. 5. Fatty acid synthesis as a function of acetyl-CoA concentration at different levels of malonyl-CoA and a fixed level of NADPH in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 1, except that the concentrations of malonyl-CoA were as follows: (A) 4 1~ (O---O), 6 WM(A-A), and 8 go (A-A); (B) 10 jtM (O-O), 20 pM (A-A), 30 pM (A-A), and 40 pM (O---O). A 5-cm cell was used for assay. Units on the ordinate are nmoles NADPH oxidized per min per mg protein.
100
80 Y 0
60
; 40
20
0
2 FATTY
4
6
ACID SYNTHETASE
e IN IJ~
FIG. 6. Fatty acid synthetase activity at an acetylCoA: malonyl-CoA ratio of 1:4 at different levels of acetyl-CoA and malonyl-CoA in 0.2 M phosphate buffer at pH 7.0. Experimental conditions were the same as those described in the legend to Fig. 4C. Concentrations of substrates were as follows: acetyl-CoA, 4 PM, malonyl-CoA, 16 pM (04); acetyl-CoA, 5 PM, malonyl-CoA, 20 KM (a---@); acetyl-CoA, 10 pM, malonyl-CoA, 40 pM (O--O); acetyl-CoA, 15 PM, malonyl-CoA, 60 PM (A-A-A); acetyl-CoA, 20 pM, malonyl-CoA, 80 pM (A-A-A); and acetyl-CoA, 30 PM, malonybCoA, 100 &M (O--O--O). The NADPH concentration was 100 PM in each assay. A l-cm cell was used for the assays as described in the Experimental Procedure. Velocity units on the ordinate are nmoles NADPH oxidized per min.
to a hydroxyl site and then to a 4’-phosphopantetheine site, a blockage of either site by the acetyl moiety would result in the inhibition of fatty acid synthesis. Our observation that at concentrations of acetyl-CoA higher than twice the concentration of malonyl-CoA a high inhibition occurs, is therefore consistent with the earlier binding studies (3, 13). The milder inhibition of fatty acid synthesis by malonyl-CoA at concentrations higher than four times the acetyl-CoA concentration observed in the present study is consistent with the fact that the enzyme is capable of synthesizing fatty acids from only malonyl-CoA and NADPH (14). In this process the malonyl moiety is decarboxylated (presumably when bound to 4’-phosphopantetheine) to yield an acetyl moiety. It is evident from our results that the availability of the binding sites or the proper moiety (acetyl or malonyl groups) is of prime importance for the maximum rate of synthesis of fatty acids. The partial blockage of the 4’-phosphopantetheine and possibly the hydroxyl site by an acetyl or a malonyl group results in the competitive inhibition observed by us. Inhibition of the synthetase activity by malonyl-CoA has also been noted by Plate et al. (5). However, the complete inhibition of the synthe-
SUBSTRATE
INHIBITION
OF FATTY
sis of fatty acids at 30 pM acetyl-CoA and reported by these 75 pM malonyl-CoA workers, was not observed by us. It appears in retrospect that Plate et al. (5) were probably working with a partially dissociated synthetase complex since their studies were carried out in the presence of low ionic-strength buffers. It has been shown by us that pigeon liver fatty acid synthetase is completely dissociated into half-molecular weight subunits in low ionic strength Tris-glycine buffer (8).
5.
6.
7.
8. 9.
REFERENCES 1. Hsu, R. Y., WASSON, G., ANDPORTER, J. W. (1965). J. Biol. Chem. 240, 3736-3746. 2. PHILLIPS, G. T., NIXON, J. E., ABRAMOVITZ, A. S., AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 357-371. 3. NIXON, J. E., PHILLIPS, G. T., ABRAMOVITZ, A. S., AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 372-379. 4. PHILLIPS, G. T., NIXON, J. E., DORSEY, J. A., BUTTERWORTH, P. H. W., CHESTERTON, C. J.,
10. 11. 12. 13.
14.
ACID SYNTHESIS
331
AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 380-391. PLATE, C. A., JOSHI, V. C., SEDGWICK, B., AND WAKIL, S. J. (1968) J. Biol. Chem. 243, 5439-5445. BUITERWORTH, P. H. W., YANG, P. C., BOCK, R. M., AND PORTER, J. W. (1967) J. Biol. Chem. 242, 3508-3516. KUMAR, S., DORSEY, J. K., AND PORTER, J. W. (1970) Biochem. Biophys. Res. Commun. 40, 825-832. KUMAR, S., MUESING, R. A., AND PORTER, J. W. (1972) J. Biol. Chem. 247, 4749-4762. LUST, G., AND LYNEN, F. (1968) Eur. J. Biochem. 7, 68-72. AYLING, J., PIRSON, R., AND LYNEN, F. (1972) Biochemistry 4, 526-532. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. CHESTERTON, C. J., BUTTERWORTH, P. H. W., AND PORTER, J. W. (1968) Arch. Biochem. Biophys. 1.26, 864-872. KATIYAR, S. S., BRIEDIS, A. V., AND PORTER, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420.