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Decarboxylation of malonyl-CoA by lactating bovine mammary fatty acid synthase
Comp. Biochem. PhysioL Vol. 90B, No. 1, pp. 179-185, 1988 Printed in Great Britain
0305-0491/88 $3.00 + 0.00 © 1988 Pergamon Press pie
DECARBOXYLATION OF MALONYL-CoA BY LACTATING BOVINE M A M M A R Y FATTY ACID SYNTHASE* SORAYA SVORONOS and SOMA KUMARt Department of Chemistry, Georgetown University, Washington, DC 20057, USA (Received 27 May 1987) Abstract--1. A pronounced malonyl-CoA decarboxylase activity of bovine mammary fatty acid synthase results in the formation of acetyl-CoA and not of triacetic acid lactone as in the reaction by yeast and pigeon liver synthase. 2. This activity is unaffected by the dissociation of the enzyme and is insensitive to its modification by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate or 2-chloroacetyl-CoA. 3. A 50% inhibition of the activity observed on the depletion of free CoA from the medium indicates that at least part of the reaction occurs only after the acylation of the enzyme with the malonyl group. 4. A parallel reaction without such a transfer also appears to occur simultaneously.
INTRODUCTION Fatty acid biosynthesis occurs by the elongation of the primer acetyl-CoA, by a sequential addition of two-carbon units derived from malonyl-CoA. Each addition step involves a condensation
strates (Lynen, 1969; K u m a r et al., 1970). We investigated the malonyl-CoA decarboxylation reaction that enables the enzyme to synthesize fatty acids at low rates even in the absence of a primer (Katiyar et al., 1974; Kresze et al., 1977). The results of these studies constitute the present communication. MATERIALS AND METHODS Fatty acid synthase The enzyme from lactating bovine mammary gland, rat liver and rat adipose was prepared by the procedure described by Strom et al. (1979) or where indicated by that of Hardie and Cohen (1978), Burton et al. (1968) and Stoops et al. (1979), respectively. Fatty acid synthase from yeast, chicken liver, and goose uropygial gland were generous gifts from Drs S. J. Wakil, S. Kumar and P. E. Kolattukudy, respectively. Assay of the different activities of synthase The overall fatty acid synthetase, crotonyl-CoA and acetoacetyl CoA reductase activities were assayed by the procedure described earlier (Strom et al., 1979; Dodds et al., 1981). For the overall activity, acetyl-CoA was used as primer. The conditions for the assay of yeast enzyme was as described by Lynen (1969). The condensing reaction was assayed by determining the rate of formation of acetoacetylCoA as described earlier (Ghayourmanesh and Kumar, 1981). Malonyl-CoA decarboxylase activity was measured by two different procedures. (1) The spectrophotometric method measured the rate of formation of acetyl-CoA by coupling it to citrate synthesis, by the procedure described by Kim and Kollattukudy (1978a) with modifications. The reaction mixture, containing 0.1 M Tris-HC1, pH8.0, 0.5 mM dithioerythritol, 10mM L-malate, 0.5 mM NAD + and 1.7 units of malate dehydrogenase was incubated for 7 min at 37°C. 1.7 units of citrate synthase was added and after l min, malonyl-CoA, 300ttM and 5/zg fatty acid synthase were added to obtain a total volume of 0.4 ml. The increase in absorbance at 340 nm observed after the first 30 seconds was recorded. It was necessary to follow this procedure rigidly in order to obtain reproducible results. (2) The standard radiochemical assay used a 10ml wheaton serum vial provided with a center well for the incubation. The reaction mixture contained 0.25 M phosphate buffer, pH 7.0, unless stated otherwise 150/zM [1,3-14C]-malonyl-
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SORAYA SVORONOS and SOMA KUMAR Table 1. Fatty acid synthetase and malonyl-CoA decarboxylase activities of the enzyme during its purification Malonyl-CoA decarboxylase Enzyme Fatty acid synthetase activity preparation activity Radioassay Spectrophotometric Particle-free cytosol 54.8 9 18 DEAE Biogel A eluant 223 23 68 Ammonium sulphate precipitate 665 80 200 UltrogeI-AcA 22 filtrate 828 101 260 The purification procedure was that of Hardie and Cohen (1978). The synthetase activity is expressed as nmol NADPH oxidized/min/mg and malonyI-CoA decarboxylase activity is expressed as nmol COLor AcCoA formed per min/mg protein.
CoA (specific radioactivity of 7 4 Ci per mol), and 5-10 pg of enzyme in a total volume of 0.2 ml. This was placed in the center well of the vial and was immediately sealed using a rubber flange. The outer compartment of the bottle contained 0.1 ml of 1 M KOH. The incubation, usually 5 rain at 37°C, was terminated by injecting 0.1 ml of 1 M H3PO 4 through the flange and the bottles were shaken gently for two hours. The 14C content of the KOH was determined by liquid scintillation counting. Counting efficiency was approximately 90%.
Dissociation of synthase Dissociation of the enzyme was achieved by dialysis against Tris (5 mM)-glycine (35 raM), pH 8.45 (Kumar et al., 1970) or against 1 mM phosphate buffer (Poulose and Kolattukudy, 1981).
Inhibition of synthase with SH reagents The enzyme (2 mg/ml) was gel-filtered through Sephadex G-75, equilibrated with deoxygenated buffer containing EDTA but no dithiothreitol. Aliquots of this enzyme solution were added to freshly prepared iodoacetamide, Nethyl-maleimide or p-hydroxymercuribenzoate of different concentrations. The mixture was kept for 30 minutes at room temperature after which the excess reagent was neutralized by the addition of dithiothreitol to obtain a final concentation of 10raM. Enzyme activities in appropriate controls were determined along with each set of experiments.
Modification of synthase with 2-ehloroacetyl-CoA This compound was prepared by the method of Kawaguchi et al. (1981) and purified by chromatography through DEAE-cellulose, equilibrated with 0.003 M HC1. Chloroacetyl-CoA was eluted by KCI gradient elution. 1.14 ml of 2.2 × 10 -7 M enzyme was reacted with a 56 molar excess of chloroacetyl-CoA for 5 minutes. Aliquots were removed for the different assays. Control reactions were carried out in parallel incubations containing amounts of HC1 and KC1 equal to that present in the aliquots of chloroacetyl-CoA modified enzyme used.
Determination of the purity of malonyl-CoA [1,3-14C]-malonyl-CoA (New England Nuclear) containing added unlabelled acetyl-CoA, was chromatographed on a polyethyleneimine-coated plate (Brinkman, MN 300). The
plate was developed with 1.0 M acetic acid, 0.6 M LiC1 and dried in a fume hood. The spots were located under UV-lamp, scraped and counted for radioactivity. In this system, malonyl-CoA is well separated from acetyl-CoA (Rf 0.35 and 0.5, respectively).
RESULTS
Copurification of fatty acid synthetase and malonylCoA decarboxylase activities The rates of the fatty acid synthetase activity a n d of the m a l o n y l - C o A decarboxylase activity, assayed by the two different methods, are shown for the enzyme p r e p a r a t i o n after each step of purification (Table 1). The difference in the decarboxylase activity as assayed by the two different procedures is und o u b t e d l y due to the differences in the conditions t h a t h a d to be employed a n d the use of m a l o n y l - C o A c o n c e n t r a t i o n c o r r e s p o n d i n g to its K m value in the radioassay. The relative constancy o f the ratio of the different activities at each step is suggestive of the decarboxylase activity being an integral part of fatty acid synthase. The purity of the final enzyme preparation h a d been established previously using several different criteria ( M a i t r a a n d K u m a r , 1974). F u r t h e r , polyacrylamide gel-electrophoresis of the native enzyme in 0.05 M p h o s p h a t e buffer (Chalberg, 1983) revealed a single b a n d . Gel-filtration t h r o u g h ultrogel AcA22 revealed the elution o f the protein in a single b a n d o f Mr 5 x 105. SDS-gel electrophoresis showeo a m a j o r protein b a n d c o r r e s p o n d i n g to Mr 250,000 a n d traces of proteins t h a t were slightly smaller in size (Chalberg, 1983). The latter are believed to be proteolytic p r o d u c t s of the synthase which are held together in the native enzyme (Stoops et al., 1978). The possibility t h a t the high m a l o n y l - C o A decarboxylase activity was artifactual a n d was due to a c o n d e n s a t i o n between m a l o n y l - C o A a n d contaminating acetyl-CoA could be ruled out. Polyethyleneimine thin-layer c h r o m a t o g r a p h y failed to detect the presence of any acetyl-CoA. Table 2 shows the two
Table 2. Fatty acid synthetase and malonyl-CoA decarboxylase activities of the synthase from various sources Malonyl-CoA decarboxylase Fatty acid synthetase activity Source activity* Tracer assay Cow mammary gland 828 88 Goose uropygial gland 950 16 Rat liver 1310 24 Rat adipose 923 32 Chicken liver 1300 18 Yeast 2500 28 *nmol of NADPH oxidized per min/mg protein.
Decarboxylation of malonyl-CoA
181
1600
ACETYLHYDROXAMATE RALONYL-
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HYOROXAMATE RF, 0.08 201 -
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8
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20 TIME (MIN)
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Fig. 1. Demonstration of the formation of acetyl-CoA on the decarboxylation of malonyl-CoA. 500pM [1,3-~4C]malonyl-CoA (specific radioactivity, 7.5 mCi per mmol) in a volume of 0.2ml was decarboxylated with 50pg of synthase for 15 min. The pH was adjusted to 7.0 with KOH and 0.6#mol of freshly prepared, neutralized hydroxylamine was added and allowed to stand at room temperature for 30 minutes. The mixture now containing acyl hydroxamates was lyophilized and extracted four times with 1 ml ice-cold ethanol. The pooled extract was concentrated under a stream of air and desalted by storage overnight at -20°C and centrifugation. The supernatant was chromatographed on Whatman No. 1 filter paper using dichloromethane: butanol: acetic acid: H20 (80:20:15:35) (Kumar and Avena, 1963). The paper was dried, cut into 1 cm sections, and the 14C-activity in each determined.
7' 80
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I 7
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activities of the enzyme from various sources. The decarboxylase activity is most pronounced in the bovine m a m m a r y synthase. As a result of this activity nearly 30% of the overall fatty acid synthetase activity is obtained in the presence of N A D P H and in the absence of acetyl-CoA (data not presented).
x
20
Formation of acetyl-CoA Evidence for the actual formation of acetyl-CoA is the high rate of reaction obtained in the spectrophotometric assay. This involved the condensation of acetyl-CoA and oxaloacetate using citrate synthase, an enzyme highly specific for both of its substrates. Additional evidence for the formation of acetyl-CoA was obtained by the analysis of the thiol esters remaining after the termination of the reaction. For this, the thiol esters were converted to hydroxamates and chromatographed. With the solvent system used acetyl-hydroxamate has an Rf of 0.35 ( K u m a r and Avena, 1963). A radioactive peak with this Rf shows the formation of this c o m p o u n d (Fig. 1). No evidence was obtained for the formation of triacetic acid lactone (Rf, 0.7) or even of methyl isoxazolone ( R f , 0.5) (Ghayourmanesh and Kumar, 1981), which would have been produced had acetoacetyl-CoA been among the reaction products.
7
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5 I 20
I q0
I 60
[MAL-CoAI (~M)-1 x 103 Fig. 2. The velocity of malonyl-CoA decarboxylation under different conditions. The standard tracer assay was used except for one variable. (a) time; (b) pH; (c) malonyl-CoA concentration. trations of up to 10 #g (data not shown) and, was linear for about 10 min (Fig. 2A). It had a pH profile (Fig. 2B) very similar to that of the synthetase activity but not of the two reductase activities (Dodds et al., 1981; Maitra and Kumar, 1974). It had a K m of 150/~M and Vmax of 320 nmol/min/mg protein (Fig. 2C).
Kinetics of the reaction
Effect of heat denaturation
U n d e r the standard conditions of the assay, the activity was linear with respect to enzyme concen-
Figure 3 shows the relative loss of the two activities as a result of heat denaturation. The nearly parallel
182
SORAYA SVORONOSand SOMAKUMAR lOOq
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i
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Fig. 3. Stability of fatty acid synthase and malonyl-CoA decarboxylase activities. Synthase was incubated at 45°C. Aliquots were withdrawn at different times and the two activities were determined under standard conditions. The spectrophotometric assay was employed for the decarboxylation reaction. Circles and triangles indicate the synthetase and decarboxylase reactions, respectively.
loss of the two activities on denaturation, or on prolonged storage, are additional indications that the two activities reside on the same protein at, most probably, the same site or closely spaced sites.
Effect of dissociation on the different reactions catalyzed by synthase Comparison of the overall fatty acid synthetase activity and some of the partial activities of the native dimeric form of the enzyme with the corresponding activities of the monomeric form are presented in Table 3. It can be seen that upon dissociation of the enzyme, all the activities are lost, or substantially diminished, except the decarboxylase activity. Upon reassociation, 90-95% of all of the original activities were recovered (Dodds et al., 1981). Similar results were obtained when the synthase was dissociated by dialysis against 1 mM phosphate, 1 mM dithiothreitol, 1 mM EDTA, pH 8 for 72 hours. It, thus, appears that the dimeric form of the enzyme is not necessary for the decarboxylase activity but it is required for not only the condensing reaction but, as reported earlier, for the reduction of acetoacetyl-CoA and crotonyl-CoA as well (Dodds et al., 1981; Maitra and Kumar, 1974).
Effect of SH-inhibitors on the activities of FAS The modification of the susceptible cysteinyl-SH of the condensing component of the cow mammary
(IODOACETAMIDE) MM
Fig. 4. Effect of modification of the enzyme using iodoacetamide at different concentrations. After its modification the enzyme was assayed for its various activities;synthetase, A - - A ; acetoacetyl-CoA reductase, O - - O ; crotonyl-CoA reductase, O--©; acetoacetyl-CoA synthetase, O - - O ; malonyl-CoA decarboxylase, m--m.
enzyme by iodoacetamide failed to produce any effect on the malonyl-CoA decarboxylase activity (Fig. 4). All of the other partial activities and the overall synthetase activity of the enzyme were completely abolished on exposure of the enzyme to ~ 3 mM iodoacetamide. Similarly, N-ethyl-maleimide at concentrations below 0.4 mM abolished all of the reactions except the decarboxylase activity. This activity remained unaffected at an inhibitor concentration as high as 6 mM. Similarly, p-hydroxymercuribenzoate of up to 0.3 mM, failed to inhibit only the decarboxylase activity (Fig. 5).
Effect of chloroacetyl-CoA 2-Chloroacetyl-CoA was reported to react specifically with the pantetheinyl-SH of the synthase from animal tissues (McCarthy et al., 1983). Yuan and Hammes (1985) found, however, that besides the pantetheinyl SH, which was completely modified, the cysteinyl SH of the condensing domain was also modified to a significant extent. The results of the assays of the various activities obtained after the enzyme was allowed to react with chioroacetyl-CoA are presented in Table 4. The decarboxylase activity was again the only one that remained unaffected. No loss of the decarboxylase activity occurred even when the enzyme was exposed to a 560 M excess of the modifying reagent.
Table 3. Effect of dissociationof the synthaseon its activities Original Activityafter Percentactivity Activity activity dissociation remaining Fatty acid synthetase Acetoacetyl-CoA reductase Crotonyl-CoA reductase Condensing reaction MalonyI-CoA decarboxylase*
540 2500 600 200 300
57 138 138 0 300
10 5 23 0 100
The enzyme was dissociated by dialysis against Tris-glycine (5 raM, 35 mM), 1 mM dithiothreitol and EDTA 0.1 mM, pH 8.4 (Kumar et al., 1970). *Spectrophotometric assay.
Decarboxylation of malonyl-CoA
183
Table 5. Effect of depletion of coenzyme A on malonyl-CoA decarboxylase reaction of fatty acid synthase Total counts in CO2 released per minute Set 1 Set 2 Control 19,820 18,560 Experimental 9,560 8,280 The synthase (6/lg, specificactivity, 590), was incubated in the center well with 300/zM [1,3J4C]-malonyl-CoA(specificradioactivity 7.5 mCi per mmol), 100 units of phosphotransacetylase and 30mM acetyl-phosphate in 0.3 ml of 0.1 M Tris-HCl, pH 6.8 (Dodds et al., 1981). Control vessels had no phosphotransacetylase. The incubation period was 5 min at 36°C. The reaction was terminated, 14CO2released, and counted as described under Methods. Under the same conditions of CoA depletion acetoacetyl-CoA reductase activity of the synthase was reduced from 2160nmol NADPH oxidized/min/mg protein to 16nmol per min/mg.
2ooq
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0-~3"
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Fig. 5. Effect of modification of the enzyme using p-hydroxymercuribenzoate at the various concentrations. After modification the various activities of the enzyme were assayed. Symbols for the different reactions are the same as in Fig. 4.
Effect o f the depletion o f CoA from the reaction mixture In order to ascertain whether the transfer of malonyl group from coenzyme A to the enzyme is a prerequisite for the decarboxylation to occur, the free coenzyme A formed as a result of transacylation, was removed from the reaction mixture using phosphotransacetylase and acetyl phosphate (Dodds et al., 1981). The results presented in Table 5 show that the removal of free C o A from the reaction mixture caused only a 50% inhibition in the malonyl-CoA decarboxylase activity. Complete scavenging of free C o A by this procedure is indicated by the total loss of acetoacetyl-CoA reductase activity. DISCUSSION A malonyl-CoA decarboxylase distinct from fatty acid synthase has been reported to be present in avian tissues and lactating rat m a m m a r y gland (Kim et al., 1979; Kim and Kolattukudy, 1978b). This enzyme appears to be cytoplasmic in the avian tissues but is mitochondrial in the rat m a m m a r y gland. The enzyme from the latter has a K m of 3 3 0 p M and a specific activity of 200-300 (Kim and Kolattukudy,
1978b). The enzyme from both sources has a mol. wt 170-190 x 103 and a p H optimum of 8.5-9.0. That the malonyl-CoA decarboxylase activity of fatty acid synthase described in this work is not due to the presence of a similar enzyme present as a contaminant can be concluded from the following observations: (1) the relative constancy of the ratio of the synthetase and the decarboxylase activities throughout purification of the enzyme to homogeneity; (2) a parallel loss of the two activities as a result of heat denaturation (Fig. 3); (3) a relatively high Vmax, similar to that of rat m a m m a r y malonyl-CoA decarboxylase, which would not be expected if it was present as a contaminant; (4) insensitivity to all SH inhibitors including p-hydroxymercuribenzoate to which the rat m a m m a r y and avian decarboxylase was particularly sensitive (Kim and Kolattukudy, 1978a); and (5) the marked difference in the p H profiles. It can be concluded that the decarboxylase activity is an inherent activity of the bovine m a m m a r y fatty acid synthase. Malonyl-CoA decarboxylase activity of fatty acid synthase has been observed earlier and this activity has been held responsible for the synthesis of fatty acids, from malonyl-CoA and N A D P H in the absence of added acetyl-CoA. Modification of the yeast enzyme with N-ethylmaleimide resulted in a parallel loss of the decarboxylase and the overall fatty acid synthetase activities (Kresze et al., 1977). However, modification of this enzyme with iodoacetamide enhanced the decarboxylase activity while simultaneously inhibiting the synthetase activity. The decarboxylase activity resulted in the production of triacetic acid lactone, as well as acetyl-CoA and acetoacetate. The kinetics of the reaction was biphasic, unlike Fig. 2A. An initial burst of CO2
Table 4. Effectof modification of synthase by chloroacetyl-CoAon its various activities Activity after Percentactivity Activity Control modification remaining Synthetase 622 12 2 AcAc-CoA reductase 2788 196 7 Crotonyl-CoA reductase 541 58 11 Condensing reaction 200 0 0 Malonyl-CoA* decarboxylase 240 240 100 Enzyme solution was treated with 56 M excess of 2-chloroacetyl-CoAin KC1 solution for 5 min and then the various activities were determined. Control activities were determined after treating the enzyme solution with the same concentration of KCI and HCI. *Spectrophotometric assay.
184
SORAYA SVORONOSand SOMAKUMAR
evolution was followed by a much slower rate of acetyl-CoA, the concentration of acetyl-CoA formed reaction which was interpreted as reflecting the rate by the decarboxylation of malonyl-CoA is necessarily of hydrolysis of acetoacetic and triacetic acids. very low and the probability of the occurrence of the Pigeon liver synthase, on the other hand, did not condensation reaction is small. produce acetyl-CoA on the decarboxylation of Fatty acid synthase from the tissues of mammalian malonyl-CoA. The enzyme bound acetyl group first species differ from that of the avian tissues in produced underwent two successive decarboxylation- possessing significant acetoacetyl-CoA and crotonylcondensation with malonyl-CoA to produce triacetic CoA reductase activities (Dodds et al., 1981; Strom acid (Katiyar et al., 1974). The conclusion from both et al., 1979) and in the production or utilization of of these studies was that decarboxylation occurred butanoyl-CoA (Abdinejad et al., 1981). These reaconly after the transfer of the malonyl group from tions can be attributed to a lack of specificity of the CoA to the pantetheinyl site of the enzyme. Our acyl transferase activity of the enzyme. Malonyl-CoA data establish that neither the pantetheinyl SH, nor decarboxylation is a reaction that is particularly even the cysteinyl SH, is involved in the decarboxy- pronounced in bovine mammary synthase which lation reaction in the absence of the condensation appears not to be characteristic of all mammalian reaction. enzymes examined. Its biochemical significance, if Two mechanisms can be postulated for the decar- any, remains to be established. boxylation of malonyl-CoA. (1) A direct decarboxylation of malonyl-CoA to acetyl-CoA without the transfer of the malonyl group to any of the binding sites of the enzyme involved in fatty acid synthesis. REFERENCES (2) Malonyl group is transferred to one of the acyl binding sites, is decarboxylated and then transferred Abdinejad A., Fisher A. M. and Kumar S. (1981) Production and utilization of butyryl-CoA by fatty acid back to CoA. Evidence suggesting the occurrence of synthetase from mammalian tissues. Arch. Biochem. Biothe latter mechanism is the severe inhibition of the phys. 208, 135-145. reaction observed when the reaction mixture is de- Anderson G. J. and Kumar S. (1987) Transacylase activity pleted of free CoA (Table 5). It is possible that failure of lactating bovine mammary fatty acid synthase. FEBS to obtain complete inhibition is at least in part due Lett. 220, 323 326. to the release of the acetyl group by hydrolysis, Aprahamian S. A., Arslanian M. J. and Wakil S. J. (1982) Comparative studies on the kinetic parameters and profreeing the binding site which enables it to accept duct analyses of chicken, rat liver and yeast fatty acid another malonyl group. Such a deacylation has been synthetase. Comp. Biochem. Physiol. 71B, 577 582. observed by us and others (Kresze et al., 1977; Burton D. N., Haavik A. G. and Porter J. W. (1968) Abdinejad et al., 1981). At the same time a decarComparative studies of rat and pigeon liver fatty acid boxylation reaction unconnected with the condensing synthetases. Arch. Biochem. Biophys. 120, 141-154. reaction is suggested by the high Km for malonyl-CoA Chalberg S. C. (1983) Photochemical cross-linking of co(150/~M) compared to its Km of 20/~M in the conenzyme A to fatty acid synthetase. Doctoral dissertation. densing reaction (Ghayourmanesh and Kumar, Georgetown University, Washington, DC. 1981). Our data suggest that both the mechanisms are Dodds P. F., Guzman M. G. F., Chalberg S. C., Anderson G. J. and Kumar S. (1981) Acetoacetyl-CoA reductase plausible and that they occur simultaneously. activity of lactating bovine mammary fatty acid synthase. An important question that arises is that when the J. biol. Chem. 256, 6282-6290. malonyl group is indeed transferred to the enzyme, which has its SH group blocked, to which group is Ghayourrnanesh S. and Kumar S. (1981) Synthesis of acetoacetyl-CoAby bovine mammary fatty acid synthase. it transferred? The most likely group is the OH F E B S Lett. 132, 231-234. group of the loading or transacylase site (McCarthy Hardie D. G. and Cohen P. (1978) Purification and physand Hardie, 1982). This should be necessarily close icochemical properties of fatty acid synthetase and acetylto the two acyl accepting SH groups as well as CoA carboxylase from lactating mammary gland. Eur. J. the decarboxylation--condensation reaction site. Biochem. 92, 25-34. Under these conditions, perhaps, even when the Katiyar S. S., Breidis A. V. and Porter J. W. (1974) Synthesis of fatty acids from malonyl-CoA and NADPH pantetheinyl-SH which normally accepts the malonyl by pigeon liver fatty acid synthetase. Arch. Biochem. group is blocked, the O-bound malonyl group can be Biophys. 162, 412-420. decarboxylated and transferred back to CoA forming acetyl CoA. Curiously, when the SH groups are not Kawaguchi A., Yoshimura T. and Okuda S. (1981) A new method for the preparation of acyl-CoA thioesters. J. blocked, no condensation appears to occur between Biochem. 89, 337-339. the acetyl group formed and another incoming mal- Kim Y. S. and Kolattukudy P. E. (1978a) Malonyl-CoA onyl group in the bovine mammary enzyme as in the decarboxylase from the uropygial gland of waterfowl: pigeon liver and yeast enzymes (Katiyar et al., 1974; purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched Yalpani et al., 1969). This is most likely due to the fatty acids. Arch. Biochem. Biophys. 190, 585-597. rapid rate of the transacylation of acetyl and malonyl groups between CoASH and the enzyme in both Kim Y. S. and Kolattukudy P. E. (1978b) Malonyl-CoA decarboxylase from the mammary gland of lactating rat. forward and reverse directions (Yuan and Hammes, Purification, properties and subcellular localization. Bio1985; Anderson, 1983). Furthermore, acetyl and malchim. Biophys. Acta 531, 187-196. onyl transacylations have been shown to be indepen- Kim S. Y., Kolattukudy P. E. and Boos A. (1979) Dual sites dent of each other and to be catalyzed by the same of occurrence of malonyl-CoA decarboxylase and their domain of the synthase from animal tissues (Yuan possible functional significance in avian tissues. Comp. and Hammes, 1985; Mikkelson et al., 1985). ConBioehem. Biophys. 62B, 443-447. sequently, in the absence of exogenously added Kresze G, B., Steber L., Oesterhelt D. and Lynen F. (1977)
Decarboxylation of malonyl-CoA Reaction of yeast fatty acid synthetase with iodoacetamide. III. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide. Eur. J. Biochem. 79, 191-199. Kumar S. and Avena R. M. (1963) Paper chromatographic separation of acetyl and fl-hydroxybutyryl hydroxamates. Anal. Biochem. 5, 265-267. Kumar S., Dorsey J. A., Muesing R. A. and Porter J. W. (1970) Comparative studies of pigeon liver fatty acid synthetase complex and its subunits. J. biol. Chem. 245, 4732-4744. Lynen F. (1969) Yeast fatty acid synthase. Methods in Enzymology XIV, 17-33. Maitra S. K. and Kumar S. (1974) Physicochemical properties of bovine mammary fatty acid synthetase. J. biol. Chem. 249, 118-125. McCarthy A. D. and Hardie D. G. (1982) Evidence that the acyl-O-esters are intermediates in the catalysis. The mechanism of rabbit mammary fatty acid synthase. FEBS Lett. 150, 181-184. McCarthy A. P., Alistair A., Hardie D. G., Santikern S. and Williams D. H. (1983) Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase. FEBS Lett. 160, 296-300. McCarthy A. D. and Hardie D. G. (1983) The multifunctional polypeptide chains of rabbit-mammary fatty acid synthase. Eur. J. Biochem. 130, 185 193. Mikkelson J., Hojrup P., Rasmussen M. M., Roepstorff P.
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and Knudsen J. (1985) Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat-mammary fatty acid synthetase. Biochem. J. 227, 21-27. Poulose A. J. and Kolattukudy P. E. (1981) Role of enoyl reductase domain in the regulation of fatty acid synthase activity by interdomain interaction. J. biol. Chem. 256, 8379-8383. Stoops J. K., Arslanian M. J., Aune K. C. and Wakil S. J. (1978) Further evidence for the multifunctional enzyme characteristic of the fatty acid synthetases of animal tissues. Arch. Biochem. Biophys. 188, 348-359. Stoops J. K., Arslanian M. J., Aune K. C., Wakil S. J. and Oliver R. M. (1979) Physicochemical studies of the rat liver and adipose fatty acid synthetases. J. biol. Chem. 254, 7418-7426. Strom K. A., Galeos W. L., Davidson L. A. and Kumar S. (1979) Enoyl-CoA reduction by bovine mammary fatty acid synthetase. J. biol. Chem. 254, 8153-8158. Wakil S. J. and Stoops J. K. (1983) Structure and mechanism of fatty acid synthetase. In The Enzymes (Edited by Boyer P. D.), 3rd edn, Vol. XVI, pp. 3-83. Yalpani M., Willecke K. and Lynen F. (1969) Triacetic acid lactone, a derailment product of fatty acid biosynthesis. Eur. J. Biochem. 8, 495-502. Yuan Z. and Hammes G. G. (1985) Elementary steps in the reaction mechanism of chicken liver fatty acid synthetase. J. biol. Chem. 260, 13532-13538.
0305-0491/88 $3.00 + 0.00 © 1988 Pergamon Press pie
DECARBOXYLATION OF MALONYL-CoA BY LACTATING BOVINE M A M M A R Y FATTY ACID SYNTHASE* SORAYA SVORONOS and SOMA KUMARt Department of Chemistry, Georgetown University, Washington, DC 20057, USA (Received 27 May 1987) Abstract--1. A pronounced malonyl-CoA decarboxylase activity of bovine mammary fatty acid synthase results in the formation of acetyl-CoA and not of triacetic acid lactone as in the reaction by yeast and pigeon liver synthase. 2. This activity is unaffected by the dissociation of the enzyme and is insensitive to its modification by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate or 2-chloroacetyl-CoA. 3. A 50% inhibition of the activity observed on the depletion of free CoA from the medium indicates that at least part of the reaction occurs only after the acylation of the enzyme with the malonyl group. 4. A parallel reaction without such a transfer also appears to occur simultaneously.
INTRODUCTION Fatty acid biosynthesis occurs by the elongation of the primer acetyl-CoA, by a sequential addition of two-carbon units derived from malonyl-CoA. Each addition step involves a condensation
strates (Lynen, 1969; K u m a r et al., 1970). We investigated the malonyl-CoA decarboxylation reaction that enables the enzyme to synthesize fatty acids at low rates even in the absence of a primer (Katiyar et al., 1974; Kresze et al., 1977). The results of these studies constitute the present communication. MATERIALS AND METHODS Fatty acid synthase The enzyme from lactating bovine mammary gland, rat liver and rat adipose was prepared by the procedure described by Strom et al. (1979) or where indicated by that of Hardie and Cohen (1978), Burton et al. (1968) and Stoops et al. (1979), respectively. Fatty acid synthase from yeast, chicken liver, and goose uropygial gland were generous gifts from Drs S. J. Wakil, S. Kumar and P. E. Kolattukudy, respectively. Assay of the different activities of synthase The overall fatty acid synthetase, crotonyl-CoA and acetoacetyl CoA reductase activities were assayed by the procedure described earlier (Strom et al., 1979; Dodds et al., 1981). For the overall activity, acetyl-CoA was used as primer. The conditions for the assay of yeast enzyme was as described by Lynen (1969). The condensing reaction was assayed by determining the rate of formation of acetoacetylCoA as described earlier (Ghayourmanesh and Kumar, 1981). Malonyl-CoA decarboxylase activity was measured by two different procedures. (1) The spectrophotometric method measured the rate of formation of acetyl-CoA by coupling it to citrate synthesis, by the procedure described by Kim and Kollattukudy (1978a) with modifications. The reaction mixture, containing 0.1 M Tris-HC1, pH8.0, 0.5 mM dithioerythritol, 10mM L-malate, 0.5 mM NAD + and 1.7 units of malate dehydrogenase was incubated for 7 min at 37°C. 1.7 units of citrate synthase was added and after l min, malonyl-CoA, 300ttM and 5/zg fatty acid synthase were added to obtain a total volume of 0.4 ml. The increase in absorbance at 340 nm observed after the first 30 seconds was recorded. It was necessary to follow this procedure rigidly in order to obtain reproducible results. (2) The standard radiochemical assay used a 10ml wheaton serum vial provided with a center well for the incubation. The reaction mixture contained 0.25 M phosphate buffer, pH 7.0, unless stated otherwise 150/zM [1,3-14C]-malonyl-
180
SORAYA SVORONOS and SOMA KUMAR Table 1. Fatty acid synthetase and malonyl-CoA decarboxylase activities of the enzyme during its purification Malonyl-CoA decarboxylase Enzyme Fatty acid synthetase activity preparation activity Radioassay Spectrophotometric Particle-free cytosol 54.8 9 18 DEAE Biogel A eluant 223 23 68 Ammonium sulphate precipitate 665 80 200 UltrogeI-AcA 22 filtrate 828 101 260 The purification procedure was that of Hardie and Cohen (1978). The synthetase activity is expressed as nmol NADPH oxidized/min/mg and malonyI-CoA decarboxylase activity is expressed as nmol COLor AcCoA formed per min/mg protein.
CoA (specific radioactivity of 7 4 Ci per mol), and 5-10 pg of enzyme in a total volume of 0.2 ml. This was placed in the center well of the vial and was immediately sealed using a rubber flange. The outer compartment of the bottle contained 0.1 ml of 1 M KOH. The incubation, usually 5 rain at 37°C, was terminated by injecting 0.1 ml of 1 M H3PO 4 through the flange and the bottles were shaken gently for two hours. The 14C content of the KOH was determined by liquid scintillation counting. Counting efficiency was approximately 90%.
Dissociation of synthase Dissociation of the enzyme was achieved by dialysis against Tris (5 mM)-glycine (35 raM), pH 8.45 (Kumar et al., 1970) or against 1 mM phosphate buffer (Poulose and Kolattukudy, 1981).
Inhibition of synthase with SH reagents The enzyme (2 mg/ml) was gel-filtered through Sephadex G-75, equilibrated with deoxygenated buffer containing EDTA but no dithiothreitol. Aliquots of this enzyme solution were added to freshly prepared iodoacetamide, Nethyl-maleimide or p-hydroxymercuribenzoate of different concentrations. The mixture was kept for 30 minutes at room temperature after which the excess reagent was neutralized by the addition of dithiothreitol to obtain a final concentation of 10raM. Enzyme activities in appropriate controls were determined along with each set of experiments.
Modification of synthase with 2-ehloroacetyl-CoA This compound was prepared by the method of Kawaguchi et al. (1981) and purified by chromatography through DEAE-cellulose, equilibrated with 0.003 M HC1. Chloroacetyl-CoA was eluted by KCI gradient elution. 1.14 ml of 2.2 × 10 -7 M enzyme was reacted with a 56 molar excess of chloroacetyl-CoA for 5 minutes. Aliquots were removed for the different assays. Control reactions were carried out in parallel incubations containing amounts of HC1 and KC1 equal to that present in the aliquots of chloroacetyl-CoA modified enzyme used.
Determination of the purity of malonyl-CoA [1,3-14C]-malonyl-CoA (New England Nuclear) containing added unlabelled acetyl-CoA, was chromatographed on a polyethyleneimine-coated plate (Brinkman, MN 300). The
plate was developed with 1.0 M acetic acid, 0.6 M LiC1 and dried in a fume hood. The spots were located under UV-lamp, scraped and counted for radioactivity. In this system, malonyl-CoA is well separated from acetyl-CoA (Rf 0.35 and 0.5, respectively).
RESULTS
Copurification of fatty acid synthetase and malonylCoA decarboxylase activities The rates of the fatty acid synthetase activity a n d of the m a l o n y l - C o A decarboxylase activity, assayed by the two different methods, are shown for the enzyme p r e p a r a t i o n after each step of purification (Table 1). The difference in the decarboxylase activity as assayed by the two different procedures is und o u b t e d l y due to the differences in the conditions t h a t h a d to be employed a n d the use of m a l o n y l - C o A c o n c e n t r a t i o n c o r r e s p o n d i n g to its K m value in the radioassay. The relative constancy o f the ratio of the different activities at each step is suggestive of the decarboxylase activity being an integral part of fatty acid synthase. The purity of the final enzyme preparation h a d been established previously using several different criteria ( M a i t r a a n d K u m a r , 1974). F u r t h e r , polyacrylamide gel-electrophoresis of the native enzyme in 0.05 M p h o s p h a t e buffer (Chalberg, 1983) revealed a single b a n d . Gel-filtration t h r o u g h ultrogel AcA22 revealed the elution o f the protein in a single b a n d o f Mr 5 x 105. SDS-gel electrophoresis showeo a m a j o r protein b a n d c o r r e s p o n d i n g to Mr 250,000 a n d traces of proteins t h a t were slightly smaller in size (Chalberg, 1983). The latter are believed to be proteolytic p r o d u c t s of the synthase which are held together in the native enzyme (Stoops et al., 1978). The possibility t h a t the high m a l o n y l - C o A decarboxylase activity was artifactual a n d was due to a c o n d e n s a t i o n between m a l o n y l - C o A a n d contaminating acetyl-CoA could be ruled out. Polyethyleneimine thin-layer c h r o m a t o g r a p h y failed to detect the presence of any acetyl-CoA. Table 2 shows the two
Table 2. Fatty acid synthetase and malonyl-CoA decarboxylase activities of the synthase from various sources Malonyl-CoA decarboxylase Fatty acid synthetase activity Source activity* Tracer assay Cow mammary gland 828 88 Goose uropygial gland 950 16 Rat liver 1310 24 Rat adipose 923 32 Chicken liver 1300 18 Yeast 2500 28 *nmol of NADPH oxidized per min/mg protein.
Decarboxylation of malonyl-CoA
181
1600
ACETYLHYDROXAMATE RALONYL-
120~
RF, 0.33
HYOROXAMATE RF, 0.08 201 -
(
80~
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8
16
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[
10
20 TIME (MIN)
2q
FILTER PAPER SECTIONS
Fig. 1. Demonstration of the formation of acetyl-CoA on the decarboxylation of malonyl-CoA. 500pM [1,3-~4C]malonyl-CoA (specific radioactivity, 7.5 mCi per mmol) in a volume of 0.2ml was decarboxylated with 50pg of synthase for 15 min. The pH was adjusted to 7.0 with KOH and 0.6#mol of freshly prepared, neutralized hydroxylamine was added and allowed to stand at room temperature for 30 minutes. The mixture now containing acyl hydroxamates was lyophilized and extracted four times with 1 ml ice-cold ethanol. The pooled extract was concentrated under a stream of air and desalted by storage overnight at -20°C and centrifugation. The supernatant was chromatographed on Whatman No. 1 filter paper using dichloromethane: butanol: acetic acid: H20 (80:20:15:35) (Kumar and Avena, 1963). The paper was dried, cut into 1 cm sections, and the 14C-activity in each determined.
7' 80
40 §
I 6
I 7
I 8
PH
q0
activities of the enzyme from various sources. The decarboxylase activity is most pronounced in the bovine m a m m a r y synthase. As a result of this activity nearly 30% of the overall fatty acid synthetase activity is obtained in the presence of N A D P H and in the absence of acetyl-CoA (data not presented).
x
20
Formation of acetyl-CoA Evidence for the actual formation of acetyl-CoA is the high rate of reaction obtained in the spectrophotometric assay. This involved the condensation of acetyl-CoA and oxaloacetate using citrate synthase, an enzyme highly specific for both of its substrates. Additional evidence for the formation of acetyl-CoA was obtained by the analysis of the thiol esters remaining after the termination of the reaction. For this, the thiol esters were converted to hydroxamates and chromatographed. With the solvent system used acetyl-hydroxamate has an Rf of 0.35 ( K u m a r and Avena, 1963). A radioactive peak with this Rf shows the formation of this c o m p o u n d (Fig. 1). No evidence was obtained for the formation of triacetic acid lactone (Rf, 0.7) or even of methyl isoxazolone ( R f , 0.5) (Ghayourmanesh and Kumar, 1981), which would have been produced had acetoacetyl-CoA been among the reaction products.
7
10
/
5 I 20
I q0
I 60
[MAL-CoAI (~M)-1 x 103 Fig. 2. The velocity of malonyl-CoA decarboxylation under different conditions. The standard tracer assay was used except for one variable. (a) time; (b) pH; (c) malonyl-CoA concentration. trations of up to 10 #g (data not shown) and, was linear for about 10 min (Fig. 2A). It had a pH profile (Fig. 2B) very similar to that of the synthetase activity but not of the two reductase activities (Dodds et al., 1981; Maitra and Kumar, 1974). It had a K m of 150/~M and Vmax of 320 nmol/min/mg protein (Fig. 2C).
Kinetics of the reaction
Effect of heat denaturation
U n d e r the standard conditions of the assay, the activity was linear with respect to enzyme concen-
Figure 3 shows the relative loss of the two activities as a result of heat denaturation. The nearly parallel
182
SORAYA SVORONOSand SOMAKUMAR lOOq
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i'
i
6C
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20
20
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60
80 1
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Fig. 3. Stability of fatty acid synthase and malonyl-CoA decarboxylase activities. Synthase was incubated at 45°C. Aliquots were withdrawn at different times and the two activities were determined under standard conditions. The spectrophotometric assay was employed for the decarboxylation reaction. Circles and triangles indicate the synthetase and decarboxylase reactions, respectively.
loss of the two activities on denaturation, or on prolonged storage, are additional indications that the two activities reside on the same protein at, most probably, the same site or closely spaced sites.
Effect of dissociation on the different reactions catalyzed by synthase Comparison of the overall fatty acid synthetase activity and some of the partial activities of the native dimeric form of the enzyme with the corresponding activities of the monomeric form are presented in Table 3. It can be seen that upon dissociation of the enzyme, all the activities are lost, or substantially diminished, except the decarboxylase activity. Upon reassociation, 90-95% of all of the original activities were recovered (Dodds et al., 1981). Similar results were obtained when the synthase was dissociated by dialysis against 1 mM phosphate, 1 mM dithiothreitol, 1 mM EDTA, pH 8 for 72 hours. It, thus, appears that the dimeric form of the enzyme is not necessary for the decarboxylase activity but it is required for not only the condensing reaction but, as reported earlier, for the reduction of acetoacetyl-CoA and crotonyl-CoA as well (Dodds et al., 1981; Maitra and Kumar, 1974).
Effect of SH-inhibitors on the activities of FAS The modification of the susceptible cysteinyl-SH of the condensing component of the cow mammary
(IODOACETAMIDE) MM
Fig. 4. Effect of modification of the enzyme using iodoacetamide at different concentrations. After its modification the enzyme was assayed for its various activities;synthetase, A - - A ; acetoacetyl-CoA reductase, O - - O ; crotonyl-CoA reductase, O--©; acetoacetyl-CoA synthetase, O - - O ; malonyl-CoA decarboxylase, m--m.
enzyme by iodoacetamide failed to produce any effect on the malonyl-CoA decarboxylase activity (Fig. 4). All of the other partial activities and the overall synthetase activity of the enzyme were completely abolished on exposure of the enzyme to ~ 3 mM iodoacetamide. Similarly, N-ethyl-maleimide at concentrations below 0.4 mM abolished all of the reactions except the decarboxylase activity. This activity remained unaffected at an inhibitor concentration as high as 6 mM. Similarly, p-hydroxymercuribenzoate of up to 0.3 mM, failed to inhibit only the decarboxylase activity (Fig. 5).
Effect of chloroacetyl-CoA 2-Chloroacetyl-CoA was reported to react specifically with the pantetheinyl-SH of the synthase from animal tissues (McCarthy et al., 1983). Yuan and Hammes (1985) found, however, that besides the pantetheinyl SH, which was completely modified, the cysteinyl SH of the condensing domain was also modified to a significant extent. The results of the assays of the various activities obtained after the enzyme was allowed to react with chioroacetyl-CoA are presented in Table 4. The decarboxylase activity was again the only one that remained unaffected. No loss of the decarboxylase activity occurred even when the enzyme was exposed to a 560 M excess of the modifying reagent.
Table 3. Effect of dissociationof the synthaseon its activities Original Activityafter Percentactivity Activity activity dissociation remaining Fatty acid synthetase Acetoacetyl-CoA reductase Crotonyl-CoA reductase Condensing reaction MalonyI-CoA decarboxylase*
540 2500 600 200 300
57 138 138 0 300
10 5 23 0 100
The enzyme was dissociated by dialysis against Tris-glycine (5 raM, 35 mM), 1 mM dithiothreitol and EDTA 0.1 mM, pH 8.4 (Kumar et al., 1970). *Spectrophotometric assay.
Decarboxylation of malonyl-CoA
183
Table 5. Effect of depletion of coenzyme A on malonyl-CoA decarboxylase reaction of fatty acid synthase Total counts in CO2 released per minute Set 1 Set 2 Control 19,820 18,560 Experimental 9,560 8,280 The synthase (6/lg, specificactivity, 590), was incubated in the center well with 300/zM [1,3J4C]-malonyl-CoA(specificradioactivity 7.5 mCi per mmol), 100 units of phosphotransacetylase and 30mM acetyl-phosphate in 0.3 ml of 0.1 M Tris-HCl, pH 6.8 (Dodds et al., 1981). Control vessels had no phosphotransacetylase. The incubation period was 5 min at 36°C. The reaction was terminated, 14CO2released, and counted as described under Methods. Under the same conditions of CoA depletion acetoacetyl-CoA reductase activity of the synthase was reduced from 2160nmol NADPH oxidized/min/mg protein to 16nmol per min/mg.
2ooq
O. 1
0-2
0-~3"
[IMHIBITORI MM
Fig. 5. Effect of modification of the enzyme using p-hydroxymercuribenzoate at the various concentrations. After modification the various activities of the enzyme were assayed. Symbols for the different reactions are the same as in Fig. 4.
Effect o f the depletion o f CoA from the reaction mixture In order to ascertain whether the transfer of malonyl group from coenzyme A to the enzyme is a prerequisite for the decarboxylation to occur, the free coenzyme A formed as a result of transacylation, was removed from the reaction mixture using phosphotransacetylase and acetyl phosphate (Dodds et al., 1981). The results presented in Table 5 show that the removal of free C o A from the reaction mixture caused only a 50% inhibition in the malonyl-CoA decarboxylase activity. Complete scavenging of free C o A by this procedure is indicated by the total loss of acetoacetyl-CoA reductase activity. DISCUSSION A malonyl-CoA decarboxylase distinct from fatty acid synthase has been reported to be present in avian tissues and lactating rat m a m m a r y gland (Kim et al., 1979; Kim and Kolattukudy, 1978b). This enzyme appears to be cytoplasmic in the avian tissues but is mitochondrial in the rat m a m m a r y gland. The enzyme from the latter has a K m of 3 3 0 p M and a specific activity of 200-300 (Kim and Kolattukudy,
1978b). The enzyme from both sources has a mol. wt 170-190 x 103 and a p H optimum of 8.5-9.0. That the malonyl-CoA decarboxylase activity of fatty acid synthase described in this work is not due to the presence of a similar enzyme present as a contaminant can be concluded from the following observations: (1) the relative constancy of the ratio of the synthetase and the decarboxylase activities throughout purification of the enzyme to homogeneity; (2) a parallel loss of the two activities as a result of heat denaturation (Fig. 3); (3) a relatively high Vmax, similar to that of rat m a m m a r y malonyl-CoA decarboxylase, which would not be expected if it was present as a contaminant; (4) insensitivity to all SH inhibitors including p-hydroxymercuribenzoate to which the rat m a m m a r y and avian decarboxylase was particularly sensitive (Kim and Kolattukudy, 1978a); and (5) the marked difference in the p H profiles. It can be concluded that the decarboxylase activity is an inherent activity of the bovine m a m m a r y fatty acid synthase. Malonyl-CoA decarboxylase activity of fatty acid synthase has been observed earlier and this activity has been held responsible for the synthesis of fatty acids, from malonyl-CoA and N A D P H in the absence of added acetyl-CoA. Modification of the yeast enzyme with N-ethylmaleimide resulted in a parallel loss of the decarboxylase and the overall fatty acid synthetase activities (Kresze et al., 1977). However, modification of this enzyme with iodoacetamide enhanced the decarboxylase activity while simultaneously inhibiting the synthetase activity. The decarboxylase activity resulted in the production of triacetic acid lactone, as well as acetyl-CoA and acetoacetate. The kinetics of the reaction was biphasic, unlike Fig. 2A. An initial burst of CO2
Table 4. Effectof modification of synthase by chloroacetyl-CoAon its various activities Activity after Percentactivity Activity Control modification remaining Synthetase 622 12 2 AcAc-CoA reductase 2788 196 7 Crotonyl-CoA reductase 541 58 11 Condensing reaction 200 0 0 Malonyl-CoA* decarboxylase 240 240 100 Enzyme solution was treated with 56 M excess of 2-chloroacetyl-CoAin KC1 solution for 5 min and then the various activities were determined. Control activities were determined after treating the enzyme solution with the same concentration of KCI and HCI. *Spectrophotometric assay.
184
SORAYA SVORONOSand SOMAKUMAR
evolution was followed by a much slower rate of acetyl-CoA, the concentration of acetyl-CoA formed reaction which was interpreted as reflecting the rate by the decarboxylation of malonyl-CoA is necessarily of hydrolysis of acetoacetic and triacetic acids. very low and the probability of the occurrence of the Pigeon liver synthase, on the other hand, did not condensation reaction is small. produce acetyl-CoA on the decarboxylation of Fatty acid synthase from the tissues of mammalian malonyl-CoA. The enzyme bound acetyl group first species differ from that of the avian tissues in produced underwent two successive decarboxylation- possessing significant acetoacetyl-CoA and crotonylcondensation with malonyl-CoA to produce triacetic CoA reductase activities (Dodds et al., 1981; Strom acid (Katiyar et al., 1974). The conclusion from both et al., 1979) and in the production or utilization of of these studies was that decarboxylation occurred butanoyl-CoA (Abdinejad et al., 1981). These reaconly after the transfer of the malonyl group from tions can be attributed to a lack of specificity of the CoA to the pantetheinyl site of the enzyme. Our acyl transferase activity of the enzyme. Malonyl-CoA data establish that neither the pantetheinyl SH, nor decarboxylation is a reaction that is particularly even the cysteinyl SH, is involved in the decarboxy- pronounced in bovine mammary synthase which lation reaction in the absence of the condensation appears not to be characteristic of all mammalian reaction. enzymes examined. Its biochemical significance, if Two mechanisms can be postulated for the decar- any, remains to be established. boxylation of malonyl-CoA. (1) A direct decarboxylation of malonyl-CoA to acetyl-CoA without the transfer of the malonyl group to any of the binding sites of the enzyme involved in fatty acid synthesis. REFERENCES (2) Malonyl group is transferred to one of the acyl binding sites, is decarboxylated and then transferred Abdinejad A., Fisher A. M. and Kumar S. (1981) Production and utilization of butyryl-CoA by fatty acid back to CoA. Evidence suggesting the occurrence of synthetase from mammalian tissues. Arch. Biochem. Biothe latter mechanism is the severe inhibition of the phys. 208, 135-145. reaction observed when the reaction mixture is de- Anderson G. J. and Kumar S. (1987) Transacylase activity pleted of free CoA (Table 5). It is possible that failure of lactating bovine mammary fatty acid synthase. FEBS to obtain complete inhibition is at least in part due Lett. 220, 323 326. to the release of the acetyl group by hydrolysis, Aprahamian S. A., Arslanian M. J. and Wakil S. J. (1982) Comparative studies on the kinetic parameters and profreeing the binding site which enables it to accept duct analyses of chicken, rat liver and yeast fatty acid another malonyl group. Such a deacylation has been synthetase. Comp. Biochem. Physiol. 71B, 577 582. observed by us and others (Kresze et al., 1977; Burton D. N., Haavik A. G. and Porter J. W. (1968) Abdinejad et al., 1981). At the same time a decarComparative studies of rat and pigeon liver fatty acid boxylation reaction unconnected with the condensing synthetases. Arch. Biochem. Biophys. 120, 141-154. reaction is suggested by the high Km for malonyl-CoA Chalberg S. C. (1983) Photochemical cross-linking of co(150/~M) compared to its Km of 20/~M in the conenzyme A to fatty acid synthetase. Doctoral dissertation. densing reaction (Ghayourmanesh and Kumar, Georgetown University, Washington, DC. 1981). Our data suggest that both the mechanisms are Dodds P. F., Guzman M. G. F., Chalberg S. C., Anderson G. J. and Kumar S. (1981) Acetoacetyl-CoA reductase plausible and that they occur simultaneously. activity of lactating bovine mammary fatty acid synthase. An important question that arises is that when the J. biol. Chem. 256, 6282-6290. malonyl group is indeed transferred to the enzyme, which has its SH group blocked, to which group is Ghayourrnanesh S. and Kumar S. (1981) Synthesis of acetoacetyl-CoAby bovine mammary fatty acid synthase. it transferred? The most likely group is the OH F E B S Lett. 132, 231-234. group of the loading or transacylase site (McCarthy Hardie D. G. and Cohen P. (1978) Purification and physand Hardie, 1982). This should be necessarily close icochemical properties of fatty acid synthetase and acetylto the two acyl accepting SH groups as well as CoA carboxylase from lactating mammary gland. Eur. J. the decarboxylation--condensation reaction site. Biochem. 92, 25-34. Under these conditions, perhaps, even when the Katiyar S. S., Breidis A. V. and Porter J. W. (1974) Synthesis of fatty acids from malonyl-CoA and NADPH pantetheinyl-SH which normally accepts the malonyl by pigeon liver fatty acid synthetase. Arch. Biochem. group is blocked, the O-bound malonyl group can be Biophys. 162, 412-420. decarboxylated and transferred back to CoA forming acetyl CoA. Curiously, when the SH groups are not Kawaguchi A., Yoshimura T. and Okuda S. (1981) A new method for the preparation of acyl-CoA thioesters. J. blocked, no condensation appears to occur between Biochem. 89, 337-339. the acetyl group formed and another incoming mal- Kim Y. S. and Kolattukudy P. E. (1978a) Malonyl-CoA onyl group in the bovine mammary enzyme as in the decarboxylase from the uropygial gland of waterfowl: pigeon liver and yeast enzymes (Katiyar et al., 1974; purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched Yalpani et al., 1969). This is most likely due to the fatty acids. Arch. Biochem. Biophys. 190, 585-597. rapid rate of the transacylation of acetyl and malonyl groups between CoASH and the enzyme in both Kim Y. S. and Kolattukudy P. E. (1978b) Malonyl-CoA decarboxylase from the mammary gland of lactating rat. forward and reverse directions (Yuan and Hammes, Purification, properties and subcellular localization. Bio1985; Anderson, 1983). Furthermore, acetyl and malchim. Biophys. Acta 531, 187-196. onyl transacylations have been shown to be indepen- Kim S. Y., Kolattukudy P. E. and Boos A. (1979) Dual sites dent of each other and to be catalyzed by the same of occurrence of malonyl-CoA decarboxylase and their domain of the synthase from animal tissues (Yuan possible functional significance in avian tissues. Comp. and Hammes, 1985; Mikkelson et al., 1985). ConBioehem. Biophys. 62B, 443-447. sequently, in the absence of exogenously added Kresze G, B., Steber L., Oesterhelt D. and Lynen F. (1977)
Decarboxylation of malonyl-CoA Reaction of yeast fatty acid synthetase with iodoacetamide. III. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide. Eur. J. Biochem. 79, 191-199. Kumar S. and Avena R. M. (1963) Paper chromatographic separation of acetyl and fl-hydroxybutyryl hydroxamates. Anal. Biochem. 5, 265-267. Kumar S., Dorsey J. A., Muesing R. A. and Porter J. W. (1970) Comparative studies of pigeon liver fatty acid synthetase complex and its subunits. J. biol. Chem. 245, 4732-4744. Lynen F. (1969) Yeast fatty acid synthase. Methods in Enzymology XIV, 17-33. Maitra S. K. and Kumar S. (1974) Physicochemical properties of bovine mammary fatty acid synthetase. J. biol. Chem. 249, 118-125. McCarthy A. D. and Hardie D. G. (1982) Evidence that the acyl-O-esters are intermediates in the catalysis. The mechanism of rabbit mammary fatty acid synthase. FEBS Lett. 150, 181-184. McCarthy A. P., Alistair A., Hardie D. G., Santikern S. and Williams D. H. (1983) Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase. FEBS Lett. 160, 296-300. McCarthy A. D. and Hardie D. G. (1983) The multifunctional polypeptide chains of rabbit-mammary fatty acid synthase. Eur. J. Biochem. 130, 185 193. Mikkelson J., Hojrup P., Rasmussen M. M., Roepstorff P.
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and Knudsen J. (1985) Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat-mammary fatty acid synthetase. Biochem. J. 227, 21-27. Poulose A. J. and Kolattukudy P. E. (1981) Role of enoyl reductase domain in the regulation of fatty acid synthase activity by interdomain interaction. J. biol. Chem. 256, 8379-8383. Stoops J. K., Arslanian M. J., Aune K. C. and Wakil S. J. (1978) Further evidence for the multifunctional enzyme characteristic of the fatty acid synthetases of animal tissues. Arch. Biochem. Biophys. 188, 348-359. Stoops J. K., Arslanian M. J., Aune K. C., Wakil S. J. and Oliver R. M. (1979) Physicochemical studies of the rat liver and adipose fatty acid synthetases. J. biol. Chem. 254, 7418-7426. Strom K. A., Galeos W. L., Davidson L. A. and Kumar S. (1979) Enoyl-CoA reduction by bovine mammary fatty acid synthetase. J. biol. Chem. 254, 8153-8158. Wakil S. J. and Stoops J. K. (1983) Structure and mechanism of fatty acid synthetase. In The Enzymes (Edited by Boyer P. D.), 3rd edn, Vol. XVI, pp. 3-83. Yalpani M., Willecke K. and Lynen F. (1969) Triacetic acid lactone, a derailment product of fatty acid biosynthesis. Eur. J. Biochem. 8, 495-502. Yuan Z. and Hammes G. G. (1985) Elementary steps in the reaction mechanism of chicken liver fatty acid synthetase. J. biol. Chem. 260, 13532-13538.