BIOCHEMICAL SOCIETY TRANSACTIONS
70
Acyl-Coenzyme A Synthetases MILTON V. PARK Department of Brewing and Biological Sciences, Heriot- Watt University, Edinburgh EHI 1H X , Scotland, U.K. The enzymes catalysing the initial stage of the 8-oxidation of fatty acids, the acyl-CoA synthetases, have been classified into four groups based on specificity. These are: the short-chain (acetyl-CoA synthetase; EC 6.2.1 .l), medium-chain (butyryl-CoA synthetase; EC 6.2.1.2) and the long-chain fatty acyl-CoA synthetase (acyl-CoA synthetase; EC 6.2.1.3), which are ATP-dependent and follow the reaction : ATP RvC02H COA + R*CO.CoA AMP+ PPi and the GTP-dependent enzyme [acyl-CoA synthetase (GDP-forming); EC 6.2.1 .lo], following the reaction: GTP+ R-COZH COA + R CO.COA GDP Pi The properties of these enzymes have been reviewed (Londesborough & Webster, 1974; Groot et al., 1976).
+
+
+
+
+
+
AMP-forming acyt-CoA synthetases Substrate specijicity. In recent years the main interests under this heading have been in identifying whether a separate propionyl-CoA synthetase exists, and whether there is more than one long-chain acyl-CoA synthetase. Differences in the specificity of the butyryl-CoA synthetases from different sources have been known for some time; the enzyme isolated from ox liver mitochondria (Mahler et a/., 1953) has a much broader specificity than that from ox heart mitochondria (Webster et a/., 1965). The variation ofenzymic activity as a function of fatty acid chain length, measured in a rat liver microsomal preparation, guggested that more than one enzyme might be involved (Pande, 1972). However, competition studies with both saturated and unsaturated fatty acids indicate that there is only the one microsomal enzyme in rat liver (Marcel & Suzue, 1972; Suzue & Marcel, 1972a,b). Groot & Van Loon (1975), using fatty acid-competition experiments, showed that, in guinea-pig heart mitochondria, most propionate activation is catalysed by acetyl-CoA synthetase, there being little evidence for a distinct propionyl-CoA synthetase. I n liver mitochondria, however, there was good evidence for an enzyme with high affinity towards propionate and some affinity towards acetate and butyrate. These observations were confirmed by the isolation of three soluble ATP-dependent acyl-CoA synthetases from guinea-pig liver mitochondria : a medium-chain acyl-CoA synthetase, a salicylateactivating enzyme, and a propionyl-CoA synthetase accepting acetate, propionate and butyrate, but with a high preference for propionate (Groot & Scheek, 1976). Mechanism. The generally accepted mechanism of action of the ATP-dependent acyl-CoA synthetases is that suggested by Berg (1956), in which an acyl-AMP intermediate is postulated. Londesborough & Webster (1 974) have reviewed the evidence for this mechanism. More recent reports have produced somewhat equivocal evidence for the acyl-AMP intermediate. Kinetic investigations using acetyl-CoA synthetase from different sources (Huang & Stumpf, 1970; Young & Anderson, 1974; Farrar &Plowman, 1975; Frenkel & Kitchens, 1977) are consistent with a mechanism in which MgATP is added first, followed by acetate, then the release of MgPPi immediately before the addition of CoA. Dedyukina et al. (1972), however, were unable to demonstrate the presence of 3H bound to protein when [3H]ATP and [14C]acetatewere incubated with the enzyme, although a 1 :1 molar ratio [14C]acetate-enzyme stable complex was formed in the presence of ATP. Anke & Spector (1975) suggested that an acetyl-enzyme was an intermediate in the overall ligase reaction after the observation of the rapid and reversible transfer of an acetyl group between CoA and dephospho-CoA in the absence of ATP or AMP. 1978
572nd MEETING, LONDON
71
O n the basis of differences in partial reactions between a purified microsomal acylCoA synthetase and the parent microsomal fraction, Bar-Tana ef a/. (1972) suggested that two activities might be present, with only one involving bound palmitoyl-AMP. In a more recent paper it was noted that although [3H]palmitate interacted with enzyme in the presence of ATP, the label only remained bound to the enzyme when relatively high ATP concentrations were present (Bar-Tana et a/., 1973). They concluded that the intermediate was either enzyme-palmitoyl-AMP or AMP-enzyme-palmitate. Molecuiarproperties. The molecular weights of acetyl-CoA synthetase from a number of sources have been reported. The enzymes from potato (Huang & Stumpf, 1970) and the mitochondria of ox heart (Londesborough et a / . , 1973), rat liver (Groot et a/., 1974) and lactating goat and bovine mammary gland (Cook et a / . , 1975; Qureshi & Cook, 1975) all had molecular weights in the range 57000-67000, although there was evidence of multiple forms of similar molecular weight in some cases (Huang & Stumpf, 1970; Qureshi & Cook, 1975). The enzymes from lactating goat and bovine mammary gland appear to be glycoproteins (Stamoudis & Cook, 1975). The enzyme from yeast is of much higher molecular weight (151000) and consists of two subunits (Frenkel & Kitchens, 1977). Rat liver microsomal acyl-CoA synthetase aggregated in solution with minimal molecular weight as determined by gel filtration and conventional sedimentation equilibrium, of about 170000, and was associated with 8.2mol of phospholipid/mol. In guanidine hydrochloride or sodium dodecyl sulphate there was partial breakdown of the molecule to give a subunit of mol.wt. 27000 and aggregates (Bar-Tana & Rose, 1973). In 75 ”/, (v/v) 2-chloroethanol the molecule completely dissociated into one polypeptide chain of mol.wt. 28000, corresponding to a catalytic unit of 168000 (Maes & Bar-Tana, 1977). The butyryl-CoA synthetase from ox liver has been reported to have a mol.wt. of 65000 by thin-layer gel Chromatography and sodium dodecyl sulphate/polyacrylamidegel electrophoresis, and to aggregate readily (Osmundsen & Park, 1975), and the enzyme from rat liver to have a mol.wt. of 47000 by gel chromatography (Groot et a/., 1974). More recent results (R. Johnston & M. V. Park, unpublished work) have indicated that the enzyme extracted from an acetone-dried powder of ox liver mitochondria in the presence of mercaptoethanol has a mol.wt. of approx. 40000, whereas when extracted from freeze-dried mitochondria a species of mol.wt. about 53000 was found, with no evidence of a 40000-mol.wt. form. In both cases molecular weights were determined by gel filtration and sedimentation equilibrium. With low concentrations, or in the absence, of mercaptoethanol o r other reducing agent, the enzyme was found to associate with itself or with bovine serum albumin to form higher-molecular-weight, enzymically active species separable by gel filtration. These observations may explain the 65000mol.wt. species mentioned above, the two different interconvertible forms of this enzyme reported by Bar-Tana et a / . (1968), and the different kinetic patterns reported for different preparations of this enzyme (Graham & Park, 1969; Bar-Tana & Rose, 1968a,b).
G DP-forming acyl-CoA synthetase Since the isolation of this enzyme (Galzigna et al., 1967), the finding of the role of phosphatidylcholine in its specificity, and the implication of 4’-phosphopantetheine as a cofactor bound to the enzyme (Rossi & Carignani, 1971), relatively little has been published o n it. Lippel & Beattie (1970) found that the GTP-dependent enzyme was absent from the outer mitochondria1 membrane and appeared to be equally associated with the matrix and with the inner membrane. Rossi & Carignani (1971) concluded that two different enzymes were present: one specific for long-chain fatty acids in the inner membrane, and the other specific for short-chain acids, probably in the matrix. These results would be consistent with those of Garland et al. (1970).
Vol. 6
72
BIOCHEMICAL SOCIETY TRANSACTIONS
O n the basis of studies of palmitate oxidation in intact uncoupled mitochondria in the presence of fluoride, Batenburg & Van den Bergh (1973) concluded that the GTPdependent enzyme does not play a n important role in the activation of fatty acids by uncoupled mitochondria. A similar conclusion was reached by Van To1 (1975). Anke, H. & Spector, L. B. (1975) Biochem. Biophys. Res. Comnzun. 67, 767-773 Bar-Tam, J. & Rose, G. (1968~)Biochem. J. 109,275-282 Bar-Tana, J. & Rose, G. (19686) Biochem. J . 109,283-292 Bar-Tana, J. & Rose, G. (1973) Biochem. J. 131,443-449 Bar-Tana, J., Rose, G. & Shapiro, B. (1968) Biochem. J. 109,269-274 Bar-Tana, J., Rose, G. & Shapiro, B. (1972) Biochem. J. 129, 1101-1107 Bar-Tana, J., Rose, G. & Shapiro, B. (1973) Biochem. J. 135,411-416 Batenburg, J. J. &Van den Bergh, S. G. (1973) Biochim. Biophys. Acta 316, 136-142 Berg, P. (1956)J. Biol. Chem. 222,991-1013 Cook, R. M., Simon, S. & Ricks, C. A. (1975)J. Agric. Food Chetn. 23,561-563 Dedyukina, M. M., Severin, E. S. & Khomutov, R. M. (1972) Biokhitniya37,862-868 Farrar, W. W. & Plowman, K. M. (1975) Znt. J. Biochem. 6,537-542 Frenkel, E. P. & Kitchens, R. L. (1977)J. B i d . Chem. 252,504-507 Galzigna, L., Rossi, C. R., Sartorelli, L. & Gibson, D. M. (1 967) J . Biol. Chem. 242,21 I 1-2 I I5 Garland, P. B., Yates, D. W. & Haddock, B. A. (1970) Biochem. J. 119,553-564 Graham, A. B. & Park, M. V. (1969) Biochem. J. 111,257-262 Groot, P. H. E. & Scheek, L. M. (1976) Biochim. Biophys. Acta441,260-267 Groot, P. H. E. & Van Loon, C . M. I. ( I 975) Biochim. Biophys. Acfa 380, 12-20 Groot, P. H. E., Van Loon, C. M. I. & Hulsmann, W. C. ( I 974) Biochim. Biophys. Acta 337, 1-12
Groot, P. H. E., Scholte, H. R. & Hulsmann, W. C. (1976) Adu. LipidReJ. 14, 75-126 Huang, K. P. & Stumpf, P. K. (1970) Arch. Biochem. Biophys. 140, 158-173 Lippel, K. & Beattie, D. S. (1970) Biochim. Biophys. Acta 218,227-232 Londesborough, J. C. & Webster, L. T. (1974) Enzymes 3rd Ed. 10,469-488 Londesborough, J. C., Yuan, S. L. & Webster, L. T. (1973) Biochem. J. 133,23-36 Maes, E. & Bar-Tana, J. (1977) Biochim. Biophys. Actn 480,527-530 Mahler, H. R., Wakil, S. J. &Bock, R. M. (1953) J. Biol. Chem. 204,453-468 Marcel, Y. L. & Suzue, G. (1972) J . Biol. Chem. 247,4433-4436 Osmundsen, H. & Park, M. V. (1975) Biochem. Soc. Trans. 3,327-329 Pande, S. V. (1972) Biochim. Biophys. Acta 270, 197-208 Qureshi, S. & Cook, R. M. (1975) J. Agric. Food Chem. 23, 563-567 Rossi, C. R. & Carignani, G. (1971) Adu. Exp. Med. B i d . 14, 147-159 Starnoudis, V. &Cook, R. M. (1975) J. Agric. Food Chem. 23,555-560 Suzue, G. & Marcel. Y. L. (1972a)J. B i d . Chem. 247, 6781-6783 Suzue, G. & Marcel, Y. L. (1972b) Biochemistry 11, 1704-1708 Van Tol, A. (1975) Mol. Cell. Biochemistry 7, 19-31 Webster, L. T., Gerowin, L. D. & Rakita, L. (1965)J. Biol. Chem. 240,29-33 Young, 0 .A. &Anderson, J. W. (1974) Biochem. J. 137,435442
The Role of Carnitine, the Carnitine Acyltransferases and the Carnitine-Exchange System R O N A R. RAMSAY Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge C B 2 1Q W, U.K. It is now well established that fatty acyl groups are transported into mitochondria by a carnitine-dependent process (Bremer, 1962; Fritz & Yue, 1963) that links the two entirely separate pools of acyl-CoA (cytosolic and intramitochondrial). It involves three steps, namely transfer of the fatty acyl moiety from CoA to carnitine, translocation of acylcarnitine from cytosol to mitochondrial matrix, and transfer of the acyl group t o mitochondria1 CoA. 1978
70
Acyl-Coenzyme A Synthetases MILTON V. PARK Department of Brewing and Biological Sciences, Heriot- Watt University, Edinburgh EHI 1H X , Scotland, U.K. The enzymes catalysing the initial stage of the 8-oxidation of fatty acids, the acyl-CoA synthetases, have been classified into four groups based on specificity. These are: the short-chain (acetyl-CoA synthetase; EC 6.2.1 .l), medium-chain (butyryl-CoA synthetase; EC 6.2.1.2) and the long-chain fatty acyl-CoA synthetase (acyl-CoA synthetase; EC 6.2.1.3), which are ATP-dependent and follow the reaction : ATP RvC02H COA + R*CO.CoA AMP+ PPi and the GTP-dependent enzyme [acyl-CoA synthetase (GDP-forming); EC 6.2.1 .lo], following the reaction: GTP+ R-COZH COA + R CO.COA GDP Pi The properties of these enzymes have been reviewed (Londesborough & Webster, 1974; Groot et al., 1976).
+
+
+
+
+
+
AMP-forming acyt-CoA synthetases Substrate specijicity. In recent years the main interests under this heading have been in identifying whether a separate propionyl-CoA synthetase exists, and whether there is more than one long-chain acyl-CoA synthetase. Differences in the specificity of the butyryl-CoA synthetases from different sources have been known for some time; the enzyme isolated from ox liver mitochondria (Mahler et a/., 1953) has a much broader specificity than that from ox heart mitochondria (Webster et a/., 1965). The variation ofenzymic activity as a function of fatty acid chain length, measured in a rat liver microsomal preparation, guggested that more than one enzyme might be involved (Pande, 1972). However, competition studies with both saturated and unsaturated fatty acids indicate that there is only the one microsomal enzyme in rat liver (Marcel & Suzue, 1972; Suzue & Marcel, 1972a,b). Groot & Van Loon (1975), using fatty acid-competition experiments, showed that, in guinea-pig heart mitochondria, most propionate activation is catalysed by acetyl-CoA synthetase, there being little evidence for a distinct propionyl-CoA synthetase. I n liver mitochondria, however, there was good evidence for an enzyme with high affinity towards propionate and some affinity towards acetate and butyrate. These observations were confirmed by the isolation of three soluble ATP-dependent acyl-CoA synthetases from guinea-pig liver mitochondria : a medium-chain acyl-CoA synthetase, a salicylateactivating enzyme, and a propionyl-CoA synthetase accepting acetate, propionate and butyrate, but with a high preference for propionate (Groot & Scheek, 1976). Mechanism. The generally accepted mechanism of action of the ATP-dependent acyl-CoA synthetases is that suggested by Berg (1956), in which an acyl-AMP intermediate is postulated. Londesborough & Webster (1 974) have reviewed the evidence for this mechanism. More recent reports have produced somewhat equivocal evidence for the acyl-AMP intermediate. Kinetic investigations using acetyl-CoA synthetase from different sources (Huang & Stumpf, 1970; Young & Anderson, 1974; Farrar &Plowman, 1975; Frenkel & Kitchens, 1977) are consistent with a mechanism in which MgATP is added first, followed by acetate, then the release of MgPPi immediately before the addition of CoA. Dedyukina et al. (1972), however, were unable to demonstrate the presence of 3H bound to protein when [3H]ATP and [14C]acetatewere incubated with the enzyme, although a 1 :1 molar ratio [14C]acetate-enzyme stable complex was formed in the presence of ATP. Anke & Spector (1975) suggested that an acetyl-enzyme was an intermediate in the overall ligase reaction after the observation of the rapid and reversible transfer of an acetyl group between CoA and dephospho-CoA in the absence of ATP or AMP. 1978
572nd MEETING, LONDON
71
O n the basis of differences in partial reactions between a purified microsomal acylCoA synthetase and the parent microsomal fraction, Bar-Tana ef a/. (1972) suggested that two activities might be present, with only one involving bound palmitoyl-AMP. In a more recent paper it was noted that although [3H]palmitate interacted with enzyme in the presence of ATP, the label only remained bound to the enzyme when relatively high ATP concentrations were present (Bar-Tana et a/., 1973). They concluded that the intermediate was either enzyme-palmitoyl-AMP or AMP-enzyme-palmitate. Molecuiarproperties. The molecular weights of acetyl-CoA synthetase from a number of sources have been reported. The enzymes from potato (Huang & Stumpf, 1970) and the mitochondria of ox heart (Londesborough et a / . , 1973), rat liver (Groot et a/., 1974) and lactating goat and bovine mammary gland (Cook et a / . , 1975; Qureshi & Cook, 1975) all had molecular weights in the range 57000-67000, although there was evidence of multiple forms of similar molecular weight in some cases (Huang & Stumpf, 1970; Qureshi & Cook, 1975). The enzymes from lactating goat and bovine mammary gland appear to be glycoproteins (Stamoudis & Cook, 1975). The enzyme from yeast is of much higher molecular weight (151000) and consists of two subunits (Frenkel & Kitchens, 1977). Rat liver microsomal acyl-CoA synthetase aggregated in solution with minimal molecular weight as determined by gel filtration and conventional sedimentation equilibrium, of about 170000, and was associated with 8.2mol of phospholipid/mol. In guanidine hydrochloride or sodium dodecyl sulphate there was partial breakdown of the molecule to give a subunit of mol.wt. 27000 and aggregates (Bar-Tana & Rose, 1973). In 75 ”/, (v/v) 2-chloroethanol the molecule completely dissociated into one polypeptide chain of mol.wt. 28000, corresponding to a catalytic unit of 168000 (Maes & Bar-Tana, 1977). The butyryl-CoA synthetase from ox liver has been reported to have a mol.wt. of 65000 by thin-layer gel Chromatography and sodium dodecyl sulphate/polyacrylamidegel electrophoresis, and to aggregate readily (Osmundsen & Park, 1975), and the enzyme from rat liver to have a mol.wt. of 47000 by gel chromatography (Groot et a/., 1974). More recent results (R. Johnston & M. V. Park, unpublished work) have indicated that the enzyme extracted from an acetone-dried powder of ox liver mitochondria in the presence of mercaptoethanol has a mol.wt. of approx. 40000, whereas when extracted from freeze-dried mitochondria a species of mol.wt. about 53000 was found, with no evidence of a 40000-mol.wt. form. In both cases molecular weights were determined by gel filtration and sedimentation equilibrium. With low concentrations, or in the absence, of mercaptoethanol o r other reducing agent, the enzyme was found to associate with itself or with bovine serum albumin to form higher-molecular-weight, enzymically active species separable by gel filtration. These observations may explain the 65000mol.wt. species mentioned above, the two different interconvertible forms of this enzyme reported by Bar-Tana et a / . (1968), and the different kinetic patterns reported for different preparations of this enzyme (Graham & Park, 1969; Bar-Tana & Rose, 1968a,b).
G DP-forming acyl-CoA synthetase Since the isolation of this enzyme (Galzigna et al., 1967), the finding of the role of phosphatidylcholine in its specificity, and the implication of 4’-phosphopantetheine as a cofactor bound to the enzyme (Rossi & Carignani, 1971), relatively little has been published o n it. Lippel & Beattie (1970) found that the GTP-dependent enzyme was absent from the outer mitochondria1 membrane and appeared to be equally associated with the matrix and with the inner membrane. Rossi & Carignani (1971) concluded that two different enzymes were present: one specific for long-chain fatty acids in the inner membrane, and the other specific for short-chain acids, probably in the matrix. These results would be consistent with those of Garland et al. (1970).
Vol. 6
72
BIOCHEMICAL SOCIETY TRANSACTIONS
O n the basis of studies of palmitate oxidation in intact uncoupled mitochondria in the presence of fluoride, Batenburg & Van den Bergh (1973) concluded that the GTPdependent enzyme does not play a n important role in the activation of fatty acids by uncoupled mitochondria. A similar conclusion was reached by Van To1 (1975). Anke, H. & Spector, L. B. (1975) Biochem. Biophys. Res. Comnzun. 67, 767-773 Bar-Tam, J. & Rose, G. (1968~)Biochem. J. 109,275-282 Bar-Tana, J. & Rose, G. (19686) Biochem. J . 109,283-292 Bar-Tana, J. & Rose, G. (1973) Biochem. J. 131,443-449 Bar-Tana, J., Rose, G. & Shapiro, B. (1968) Biochem. J. 109,269-274 Bar-Tana, J., Rose, G. & Shapiro, B. (1972) Biochem. J. 129, 1101-1107 Bar-Tana, J., Rose, G. & Shapiro, B. (1973) Biochem. J. 135,411-416 Batenburg, J. J. &Van den Bergh, S. G. (1973) Biochim. Biophys. Acta 316, 136-142 Berg, P. (1956)J. Biol. Chem. 222,991-1013 Cook, R. M., Simon, S. & Ricks, C. A. (1975)J. Agric. Food Chetn. 23,561-563 Dedyukina, M. M., Severin, E. S. & Khomutov, R. M. (1972) Biokhitniya37,862-868 Farrar, W. W. & Plowman, K. M. (1975) Znt. J. Biochem. 6,537-542 Frenkel, E. P. & Kitchens, R. L. (1977)J. B i d . Chem. 252,504-507 Galzigna, L., Rossi, C. R., Sartorelli, L. & Gibson, D. M. (1 967) J . Biol. Chem. 242,21 I 1-2 I I5 Garland, P. B., Yates, D. W. & Haddock, B. A. (1970) Biochem. J. 119,553-564 Graham, A. B. & Park, M. V. (1969) Biochem. J. 111,257-262 Groot, P. H. E. & Scheek, L. M. (1976) Biochim. Biophys. Acta441,260-267 Groot, P. H. E. & Van Loon, C . M. I. ( I 975) Biochim. Biophys. Acfa 380, 12-20 Groot, P. H. E., Van Loon, C. M. I. & Hulsmann, W. C. ( I 974) Biochim. Biophys. Acta 337, 1-12
Groot, P. H. E., Scholte, H. R. & Hulsmann, W. C. (1976) Adu. LipidReJ. 14, 75-126 Huang, K. P. & Stumpf, P. K. (1970) Arch. Biochem. Biophys. 140, 158-173 Lippel, K. & Beattie, D. S. (1970) Biochim. Biophys. Acta 218,227-232 Londesborough, J. C. & Webster, L. T. (1974) Enzymes 3rd Ed. 10,469-488 Londesborough, J. C., Yuan, S. L. & Webster, L. T. (1973) Biochem. J. 133,23-36 Maes, E. & Bar-Tana, J. (1977) Biochim. Biophys. Actn 480,527-530 Mahler, H. R., Wakil, S. J. &Bock, R. M. (1953) J. Biol. Chem. 204,453-468 Marcel, Y. L. & Suzue, G. (1972) J . Biol. Chem. 247,4433-4436 Osmundsen, H. & Park, M. V. (1975) Biochem. Soc. Trans. 3,327-329 Pande, S. V. (1972) Biochim. Biophys. Acta 270, 197-208 Qureshi, S. & Cook, R. M. (1975) J. Agric. Food Chem. 23, 563-567 Rossi, C. R. & Carignani, G. (1971) Adu. Exp. Med. B i d . 14, 147-159 Starnoudis, V. &Cook, R. M. (1975) J. Agric. Food Chem. 23,555-560 Suzue, G. & Marcel. Y. L. (1972a)J. B i d . Chem. 247, 6781-6783 Suzue, G. & Marcel, Y. L. (1972b) Biochemistry 11, 1704-1708 Van Tol, A. (1975) Mol. Cell. Biochemistry 7, 19-31 Webster, L. T., Gerowin, L. D. & Rakita, L. (1965)J. Biol. Chem. 240,29-33 Young, 0 .A. &Anderson, J. W. (1974) Biochem. J. 137,435442
The Role of Carnitine, the Carnitine Acyltransferases and the Carnitine-Exchange System R O N A R. RAMSAY Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge C B 2 1Q W, U.K. It is now well established that fatty acyl groups are transported into mitochondria by a carnitine-dependent process (Bremer, 1962; Fritz & Yue, 1963) that links the two entirely separate pools of acyl-CoA (cytosolic and intramitochondrial). It involves three steps, namely transfer of the fatty acyl moiety from CoA to carnitine, translocation of acylcarnitine from cytosol to mitochondrial matrix, and transfer of the acyl group t o mitochondria1 CoA. 1978
