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14. Fatty Acyl-CoA Synthetases
Fatty A cy I- CoA Synthetases JOHN C. LONDESBOROUGH
LESLIE T. WEBSTER, JR.
I. Introduction . . . . . . . . . . . A. Scope of This Chapter . . . . . . . B. Distribution and Isolation of Acid:CoA Ligases . 11. Molecular Properties of Acetate:CoA Ligase (AMP) . . 111. Catalytic Properties of the Fatty Acid:CoA Ligases (AMP) A. General Considerations . . . . . . . B. Acetate:CoA Ligase (AMP) . . . . . . C. Medium Chain Fatty Acid:CoA Ligases (AMP) , D. Long Chain Fatty Acid:CoA Ligase (AMP) . . E. Conclusions . . . . . . . . . .
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469 469 470 474 475 475 477 483 485 487
I. Introduction
A. SCOPE OF THIS CHAPTER The fatty acyl-CoA synthetases include the ATP-dependent fatty acid :CoA ligases (AMP) and the GTP-dependent fatty acid :CoA ligases (GDP), which catalyze reactions ( 1 ) and (2), respectively. Me¶+
+ ATP’- + COASH RCO*SCoA+ AMPa- + PP;’Met+ RCO.SCoA + GDPS- + PipRCOO- + GTP4- + COASH RCOO-
(1) (2)
The reader is referred to Jenck’s previous review of fatty acid activation ( 1 ) . In the present chapter, emphasis is placed on more recent work, particularly that concerning the short, medium, and long chain fatty 1. W. P. Jencks, “The Enzymes,” 2nd ed., Vol. 6, p. 373, 1962. 469
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER,
JR.
acid: CoA ligases (AMP). Information about other acyl-CoA synthetases, including the medium-long chain fatty acid:CoA ligase (GDP) of Rossi and co-workers ( 2 ) is generally much less complete but is summarized, together with distribution data and isolation methods, in Section 1,B. Rossi and Gibson’s enzyme is mechanistically related to the succinate: CoA ligases (ADP and GDP) which are treated in Chapter 18 (3).
B. DISTRIBUTION AND ISOLATION OF ACID:CoA LIGASES 1. Acetate:CoA Ligase ( A M P )
The acetate:CoA Ligase (AMP) (EC 6.2.1.1) enzyme has been purified 20-30-fold from several new sources including ox brain, Euglena gracilis, and Aspergillus niger (4-6) ; i t remains to be demonstrated in heterotrophic bacteria. Acetyl-CoA synthetase probably has a bimodal distribution in mammalian cells, being found in both mitochondria1 and supernatant fractions of rat liver, kidney, heart, and adipose tissue ( 7 ) . The most highly purified enzyme has been obtained from ox heart mitochondria by two different methods. Unstable, aggregating, but niicrocrystalline and apparently homogeneous material with a specific activity of 35 pmoles/min/mg a t 37” was prepared earlier by Webster (8).Recently, more stable but noncrystalline material of higher specific activity (66 pmoles/min/mg) was obtained by a modification in which a pH fractionation and neomycin step was substituted for negative absorption with alumina Cy gel (9). Sharkova has purified acetyl-CoA synthetase from rabbit heart by the earlier method to a specific activity of 12 pmoles/min/mg at 37” (10). 2. Medium Chain Fatty A c i d : C o A Ligases ( A M P ) The medium chain fatty acid:CoA ligasc (AMP) (EC 6.2.1.2) activity was first partially purified from beef liver mitochondria by Mahler et al. 2. L. Galzigna, C. R. Rossi, L. Sartorelli, and D. M. Gibson, JBC 242, 2111 (1967). 3. W. Bridger, Chapter 18, this volumc. 4. G. A. Rao, I. A. Hansen, and B. K. Bnchhawat, J . Sci. Znd. Res. Sect. C 20, 284 (1961). 5 . E. Ohrnann, BBA 82, 325 (1964). 6. V. K. Shah and C. V. Ramakrislinan, Euzymologin 26, 53 (1963). 7. C. Barth, M. Sladek, and K. Decker, BRA 248, 24 (1971). 8. L. T. Webster, Jr., “Methods in Enzymology,” Vol. 13, p. 375, 1969. 9. J. C. Londesborough, S. L. Yuan, and L. T. Webster, Jr., BJ 133, 23 (1973). 10. E. V. Sharkova, Biokhimiya 33, 792 (1968); Biochemistry ( U S S R ) 33, 648 (1968).
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(11) and since reported in a large variety of mammalian tissues and higher plants ( 1 ) . Newer sources include yeast and pseudomonads (19, IS). Mahler’s fraction was active with a variety of aliphatic and aromatic carboxylic acids (Section III,C,l) . Several different medium chain fatty acyl-CoA synthetases may occur in the same subfraction of a mammalian tissue. The hexanoate:CoA ligase (AMP) activity of beef liver mitochondria has been resolved by Killenberg et al. (14) into two fractions, both of which can activate benzoate but only one of which, purified 30fold, can activate salicylate or p-aminosalicylate. This source also contains the medium-long chain fatty acid:CoA ligase (GDP) ( 8 ) . Bar-Tana et al. have resolved the butyrate:CoA ligase (AMP) activity from Mahler’s “Fraction C” into two subfractions which may, however, be derived from a single protein (15).Again, crude extracts of dog kidney mitochondria have both butyryl-CoA and benzoyl-CoA synthetase activities, but a fraction which activates butyrate, but not benzoate, can be obtained from them (16). The substrate specificity of medium chain fatty acid:CoA ligases (AMP) may also vary with the tissue of origin. Thus, a n enzyme has been purified from beef heart mitochondria to a specific activity of 3.3 pmoles butyryl-CoA/min/mg a t 37” which, unlike related enzymes in beef liver mitochondria (11, 1 4 ) , does not activate aromatic fatty acids or aliphatic acids with more than seven carbon atoms (17).To date, no acid :CoA ligases (AMP) have been isolated which exclusively activate aromatic carboxylic acids. Plants may contain such enzymes [e.g., the CoA ligase (AMP) activity observed with cinnamic acids in extracts of spinach leaves, peas, runner beans, and parsley cell cultures], but their substrate specificities remain to be determined (18, 19).
3. Long Chain Fatty Acid:CoA Ligase ( A M P )
The long chain fatty acid:CoA ligase (AMP) (EC 6.2.1.3) type of activity has been found in particulate subfractions of many mammalian 11. H. It. Mahlcr, S. J. Wakil, and R. M. Bock, JBG 204, 453 (1953). 12. T. J. Trust and N. F. Millis, J. Bucten’ol. 104, 1397 (1970). 13. T. J. Trust and N. F. Millis, J. Bacterial. 105, 1216 (1971). 14. P. G. Killcmberg, E. D. Davidson, and L. T. Webster, Jr., Mol. Pharm. 7, 260 (1971). 15. J. Bar-Tana, G. Rosc, and B. Shapiro, BJ 109, 269 (1968). 16. L. T. Webster, Jr. and Z. R. Vlahcevic, unpublished observations. 17. I,. T. Wcbster, Jr., L. D. Gerowin, and L. Rakita, JBC 240, 29 (1965). 18. E. Walton and V. S. Butt, Phytochemktry 10, 295 (1971). 19. K. Hahlbrock and H. Griseback, FEBS Lelt. 11, 62 (1970).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, JR.
tissues (see, e.g., refs. 20 and 21) and has been reported in pseudomonads (IS), yeast ( 2 2 ) , and Bacillus megaterium (23); the activity is inducible in Escherichia coli (24, 26). The microsomal activities arc difficult to solubilize ; the microsomal enzyme of hamster intestinal mucosa may even occur as part of a glyceride synthetase complex containing a t least four different polypeptide chains ( 2 6 ) .Marcel and Suzue (27) have presented kinetic evidence that a single enzyme activates most, if not all, saturated and unsaturated long chain fatty acids in rat liver microsomes (Section II1,D). Bar-Tana et al. have solubilized palmitoyl-CoA synthetase in apparently quantitative yield from rat liver microsomes by treatment with sodium deoxycholate after their extraction with dry organic solvents (28). The extracted material was purified another 14-fold to a specific activity of 0.25 pmole/min/mg a t 37". Specific activity was constant throughout a single protein peak observed by gel filtration; a molecular weight of 250,000 was estimated under conditions where detergent was not completely removed. The activity of this preparation was increased by a great variety of detergents, although these had little effect on intact microsomes. In contrast, the soluble long chain acyl-CoA synthetase partially purified by Massaro and Lennarz from the 78,000-g supernatant of B . megaterium was not activated by any of many detergents tested and was severely inhibited by somc (29).The last enzyme requires ATP, but it has not been shown whether the nucleotide product is AMP or ADP. 4. Medium-Long Chain Fatty A r i d : P o A Ligase ( G D P ) Rossi and co-workers have obtained two partially purified preparations with GTP-dependent fatty acyl-CoA synthctase activity from rat liver mitochondria. Activity wit11 C,-C,, fatty acids was purified from acetonctreated mitochondria ( X I ) , whereas a preparation equally active with 20. S. V. Pande and J. F. Mead, BRA 152, 636 (1968). 21. K. Lippel and D. S. Henttir, BBA 218, 227 (1970). 22. %. Duvnjnk, J. M. Lebeault, B. Rorlie, and E. Azoulny, BBA 202, 447 (1970). 23. W. J. Lennarz, BBA 73, 335 (1963). 24. G. Wrrks, M. Shapiro, li. 0. Burns, and S. J. Wakil, J . Bncleriol. 97, 827 ( 1969). 25. P. Ovcrsth, G . Pnuli, nnd 11. IJ. Srhnirrr, Rur. J. Biochem. 7, 559 (1969). 26. G. A. Rao and J. M. Jolinston, BBA 125, 465 (1966). 27. Y. L. Marcel and G. Suzuc, JBC 247, 4433 (1972). 28. J. Bar-Tann, G. Rose, nnd n. Stinpiro, BJ 122, 353 (1972). 29. E. J. Massaro and W. J. Lennarz, Biochemistry 4, 85 (1965). 30. C. R. Rossi and D. M. Gibson, JUG' 239, 1694 (1964).
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both butyrate and oleate (0.1 pmoleJmin/mg a t 38”) was obtained from mitochondria not exposed to acetone ( 8 ) . Both preparations were free from succinyl-CoA synthetase and ATP-dependent acyl-CoA synthetases. Extraction with acetone of the preparation active with long chain substrates removed lipid and caused a 70% loss of both butyryl-Coh and oleoyl-CoA synthetase activities ( 3 1 ) . However, acetone extraction of sonicated mitochondria decreased only the long chain acyl-CoA synthetase but not the butyryl-CoA synthetase activity. I n both cases, addition of lecithin largely restored the activities lost during acetone treatment. The authors suggested that the different specificity of the isolated GTP-dependent enzymes for fatty acid substrates results from their different lecithin contents, but it is not yet clear if the enzymes are identical in other respects. The long chain enzyme was estimated to have a molecular weight of 20,000 by gel filtration ( 2 ) . Michaelis constants were oleate, 2.1 mM; palmitate, 3.3 mM; butyrate, 0.22 m M ; octanoate, 0.2 mM; CoA, 3.3 m M ; and GTP, 4.3 mM. The enzyme was completely inhibited by 2 mM F- and 70% inhibited by 5 mM phosphate, whereas these reagents do not inhibit long chain fatty acid:CoA ligases (AMP). Rossi et al. have now shown that the GTP-dependent enzyme contains 1 mole of 4’-phosphopantetheine/2O1000g, which is essential for activity (32). In this respect also, the enzyme resembles succinate:CoA ligase (GDP) ( 3 ) ,and differs from the fatty acid:CoA ligases (AMP) in which such a cofactor has not been demonstrated. 5. Other Related Acid:CoA Ligases
A distinct oxalic acid:CoA ligase (AMP) occurs in seeds of Lathyrus sativa (33), and lactyl-CoA synthetase activity has been found in E . coli ( 3 4 ) . A specific propionyl-CoA synthetase has been purified from sheep liver ( 3 5 ) . Acetoacetyl-CoA synthetase activity, first reported in pigcon liver by Stern and Ochoa ( 3 6 ) , is present in a wide variety of tissues including rat liver (3’7). An analogous, but GTP-linked enzyme occurs in mitochondria from brown adipose tissue and requires 4’-phos31. I,. S:irtordli, I,. Galzigna, C. R. Rossi, and D. M. Gibson, BBRC 26, 90 (1967). 32. C. R. Rossi, A. Alexandre, L. Galzigna, L. Sartorelli, and D. M. Gibson, JBC 245, 3110 (1970). 33. G. A. R. Johnston and H. J. Lloyd, Aust. J . Biol. Sei. 20, 1241 (1967). 34. R. E. Mcgraw, H. C. Reeves, and S. J. Ajl, J . Bacterial. So, 984 (1965). 35. S. B. Latimer, Ph.D. Dissertation, Univeraity of North Carolina, Raleigh, 1967. 36. J. R. Stcrn and S. Ochoa, JBC 191, 161 (1951). 37. J. R. Stern, BBRC 44, 1001 (1971).
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JOHN C. LONDESBOROUGH AND LESLIE T. WBSTER, JR.
phopantetheine (38).A possibly specific cholic acid: CoA ligase (AMP) is present in the microsomes of guinea pig and rat (39). The glutarylCoA synthetase from pigeon liver and dog muscle is cqually active with ATP, GTP, and ITP ; nucleotide diphosphate and inorganic phosphate are formed (40). Glutaryl-CoA synthctase activity is also found in Pseudomonas fluorescens and Rhodopseudomoms spheroides (41, @) .
It. Molecular Properties of Acetate :CoA Ligase (AMP)
Few of the enzymes listed in Section I,B have been purified sufficiently to permit studies of their molecular propcrtics and only acetyl-CoA synthetase from heart mitochondria has been examined in any detail. Gel filtration experiments conducted with protein concentrations over a 1000-fold range indicated a constant molecular weight of 57,000k 3500 for the high specific activity preparations of acetyl-CoA synthetase from ox heart mitochondria (9). With the essentially homogeneous but unstable enzyme prepared earlier, molecular weights estimated by sedimentation equilibrium ranged from 30,000 a t the meniscus to greater than 100,OOO a t the base, and depended on the particular preparation and its age (4.9).Two peptides in the 27,000to 30,000molecular weight region can be demonstrated by sodium dodecyl sulfate (SDS) disc gel electrophoresis after the protein is oxidized with performic acid, whereas a broad band appears in this region after the protein is reduced in SDSmercaptoethanol (4).Whether such peptides in fact represent nonidentical subunits of the native protein as described for tryptophanyltRNA synthetase from beef pancreas (45) remains to be established. The amino acid analysis of the highest specific activity preparations was unremarkable and accounted for 93.5% of the dry weight (9). No p-alanine, amino sugars, neutral sugars, or sialic acids could be demonstrated. By dry weight analysis the extinction coefficient a t 280 nm is 1.41 mg-’ cm2. The ultraviolet spectrum displays the usual peak a t 280 38. C. R. Roe& Z. Drahota, A. Alexandre, and N. Siliprandi, Abstr. FEBS Meet., 6th, 1969 p. 84 (1969). 39. W. H. Elliott, “Methods in Enzymology,” Vol. 5, p. 473, 1962. 40. G. K. K. Menon, D. L. Friedman, and J. R. Stern, BBA 44, 375 (19eo). 41. Y . Nishizuka, S. Kuno, and 0. Hapaishi, BBA 43, 357 (1960). 42. S. Lartillot, J. Dedreux, and C. Baron, Bull. SOC.Chim. B i d . 47, 919 (1965). 43. L. T. Webster, Jr., JBC 2a0, 415 (1965). 44. L. T. Webster, Jr. and L. Aldwin, unpublished observations (1973). 45. E. C. Preddie, JBC 244, 3958 (1969).
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nm and a rather high 280/260 absorption ratio (1.9-2.0 a t pH 8 ) . The absorptions a t 294.4 nm and 280 nm in 0.1M KOH are entirely consistent with the tyrosine and tryptophan contents. Fully active preparations do not therefore appear to contain significant amounts of nucleotides or pantothenate derivatives. The rabbit heart enzyme appears homogeneous in the ultracentrifuge with a sedimentation coefficient of 3.86 (10) as compared to values of s20,wranging from 3.5 to 4.8 for different preparations of the ox heart enzyme (9, 43). 111. Catalytic Properties of the Fatty Acid:CoA Ligares (AMP)
A. GENERAL CONSIDERATIONS In addition to the overall reaction, many fatty acid:CoA ligases (AMP) catalyze several partial reactions. The synthesis of hydroxamates from fatty acids, ATP, and hydroxylamine, and the synthesis of ATP from acyl adenylate and PPi are Mg-dependent but CoA-independent. The synthesis of acyl-CoA’s from acyl adenylates and CoA is Mgindependent. Berg therefore proposed that the overall reaction proceeds via an acyl adenylate intermediate (46) : M#+
+ RCOOH + E E.RCO.AMP + PPi E.RCO*AMP+ COA+ E + RCO-COA + AMP ATP
(a)
(b) This mechanism was supported by Boyer’s finding of an IRO exchange between fatty acid and AMP as well as by other exchange reactions discussed by Jencks (1). It has since been established that enzyme-bound acetyl adenylate and butyryl adenylate can be made from ATP, acetate, or butyrate and large amounts of the respective activating enzyme under appropriate conditions (Sections II1,B and C) . However, while acyl adenylates are good substrates for most, if not all, fatty acid:CoA ligases, it is far from clear whether they are obligatory intermediates in the overall reaction. Particular attention will be paid to this point in the remainder of the review. Investigations of the mechanisms of the fatty acyl-CoA synthetases are still hindered by unsatisfactory assay procedures. Acetyl-CoA synthetase activity can be monitored by coupling the reaction with those of citrate synthase and malate dehydrogenase, thereby allowing the con46. P. Berg, JBC 222, 991 (1956).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
tinuous measurement of NADH production spectrophotometrically (47, 48). However, components of the coupled system have been reported to interfere with acetyl-CoA synthetase activity (49, 5 0 ) . The sensitivity of methods using the thioester absorption a t 232 nm (49, 50) is limited by the high blank absorption a t even moderate CoA and A T P concentrations. Assays for free CoA thiol or acyl-CoA (c.g., refs. 15 and 28) have been discontinuous except recently (see ref. 7 8 ) and suffer from the usual drawbacks of poor sensitivity and accuracy. However, thc differential extraction of radioactively labeled free acids from acyl-CoA (e.g., refs. 14 and 2 7 ) , while laborious, ran give data of high quality with the more lipophilic fatty acids. Under the conditions usually used to assay thesc enzymes (pH > 7, excess Mg2+ions) more than 95% of the total A T P is present as MgATY2which is probably the true substrate. It is not known whether the enzymes can distinguish between the uncomplexed and metal chelate forms of CoA and its esters. Little information about the strength of these complexes is available. The shift of the titration curves of 8hydroxyquinoline with MgCI, in the presence and absence of CoA ran be accounted for by the formation of MgCoA with an apparent stability constant of 250 M-l in 0.24 M tris CI, p H 7.7 a t 25" ( 5 1 ) .Approximately 50% of the total CoA is therefore chelated a t a free Mgz+ concentration of 4 mM. The interpretation of kinetic data for the fatty acid activating enzymes is complicated by these additional equilibria. By comparison of the equilibria of the citrate lyase and citrate synthase reactions a t pH 7.2, 25", and p. = 0.1 M , Tate and Datta ( 5 2 ) obtained a value of -6.8 kcal/mole for the free energy of hydrolysis (AG',) of acetyl-CoA to CoA and acetate anion. This is in good agreement with the value of -7.7 kcal/mole which Jencks et d.estimated from a study of the isomerization of mercaptopropyl acetate a t pH 7.0, 39", and p = 0.3M ( 5 3 ) . All the acyl-CoA synthetase reactions are therefore expected to be freely reversible. For acetyl-CoA synthetase and the medium chain enzyme equilibrium constants close to unity have been observed (11, 47, 5 4 ) , but the effects of temperature, ionic strength, and 47. P. Hcle, JBC 206, 671 (1954). 48. D. J. Pearson, BJ 95, 23c (1965). 49. W. W. Farrar and F. M. Ploughman, Fed. Proc., Fed. Amer. SOC.Ezp. Biol.
29, 425 (1970). 50. W. W. Farrar, personal communication. 51. J. C. Londesborough, unpublished observations. 52. S. S. Tate and S. P. Datta, BJ 94, 470 (1965). 53. W. P. Jencks, S. Cordes, and J. Carriuolo, JBC 235, 3608 (1960). 54. M. E. Jones, Fed. Proc., Fed. Amer. SOC. Exp. Biol. 12, 708 (1953).
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metal ion concentration have not been studied. This information is eventually required for comparison with kinetic data. B. ACETATE :CoA LIGASE (AMP) 1. Substrates and Inhibitors Besides acetate, propionate, and acrylate ( 4 7 , 5 5 ) ,the ox heart enzyme activates isobutyrate, fluoroacetate, and probably formate (66). Rates for the last three substrates are far lower than that for acetate. Evidence that formate is a substrate is indirect; it supports a CoA-dependent breakdown of ATP. Net thiol disappearance was not shown, presumably because formyl-CoA was unstable under the assay conditions ehployed. The ox heart enzyme appears quite specific for adenine nucleotides ( 5 6 ) . At 3.6 mM, dATP gives approximately 70% the rate obtained with ATP. Neither glutathione nor pantetheine can substitute for CoA as the acyl acceptor. However, with the yeast preparation, Gunther and Mautner have shown that seleno-coenzyme A can act as both a substrate and a competitive partial inhibitor of coenzyme A ( 5 7 ) . At sufficiently high concentrations, many fatty acid substrates inhibit the rate of acyl-CoA formation (55). Acetyl-CoA synthetase may also be inhibited by certain buffers. We have recently found that the decrease in rate in tris buffer below pH 8 does not occur in piperazine-l\r,ZV’bis [ethanesulfonic acid] (PIPES) buffer and cannot be explained by changes in Michaelis constants (Table I). The inhibition by tris a t TABLE I OF BUFFER AND pH ON THE MICHAELIS CONSTANTS AND TURNOVER EFFECT NUMBER IN THE FORWARD REACTION FOR ACETYL-COA SYNTAETASE FROM Ox HEART MITOC~ONDRIA~ ~~
Parameter
TN (moles/min/ 270 (230 at 57,000 g enzyme) 20 mM K+) 0.35 K , ATP (mM) 0.22 K,,, acetate (mM) 0.22 K , CoA (mM) (I
~~
Tris, pH 8.3 PIPES, pH 7.8 (200 mM K+) (20 mM K+) 230
-
PIPEX3, pH 6.7 TI%, pH 6.7 (200 m M K+) (200 mM K+) 280
0.10 0.14 1.2
32 0.017 c2.0 0.28
30”, 5 mM MgC12, 100 mM tris or 75 m M PIPES.
55. F. Campagnari and L. T. Webster, Jr., JBC 238, 1628 (1963). 56. J. C. Londesborough and L. T. Webster, Jr., unpublished observations. 57. W. H. H. Gunther and H. G . Mautner, unpublished observations cited by H. G. Mautner, “Methods in Enzymology,” Vol. 18A, p. 338,1970.
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JOHN C. LONDESBOROUGH AND LESLIE T. W'EBSTER, J R .
neutral pH is not undcrstood. PossMy the interaction hctwccn enzyme and tris cation, which at higher pH activates the cnzymc in an analogous fashion to XH,' and K', changcs to tlic inhibitory pattern characteristic of Na' (Section III,B,3). Thr pA of the change in maximum wloeity in tris is about 7.3; tliiis, an ionization of the enzyme, ratlicr than the tris (pk' 8.3 a t 30°),appears rcsponsiblc. 2. F o r m t i o n of Enzynie-Bound Acetyl Adenylnte
As discusscd prcviously by .Jencks ( 1 ) initial failures to dcmoiistrate enzymic formation of ncctyl adcnylatc constituted a major drawl)ack to Berg's proposcd mechanism. This objcction was later remorrd whcn Wehster rcportcd tlic isolation of cnzymc-bound acctyl adcmylatc by gel filtration of rcaction mixturcs containing [l'C] acctatc, ATP-lIg, and substratc quantities of highly purified acctyl-CoA synthetasc from ox heart (58). As cxpcctcd, thc cnzyme-1)ound species was also synthcsizcd from acetyl-CoA and AMP arid rcactcd with CoA to form acetyl-CoA. This was the first observation of an isolatcd cnzyinc-hound spccics for any of thc twid:acccptor ligascs (AJIP) , and thc sanic approach was subsequently utilizcd to isolate cnzymc-bound aminoacyl adcnylatcs from a variety of amino acid-tRSA synthctasr systems (59). Howcvcr, whcthcr cnzymc-hound acrtyl adcnylatr is an obligatory intermediate in thc acetyl-CoA synthctasc rcaction is unccrtain, and yields of this complex from suhstratrs of thc partial reactions have varied markcdly dcpcnding on tlic preparation of cnzymc. Higher yicltls wcrc obtained earlirr with prcparations of variahlc stability and purity (43, 58, 60) ; the highest was found with a partially purificd cnzyiiic which had not been subjcctcd to gcl filtration during purification (60).\\'it11 the recent enzyme preparations, which are more stable and catalytically active, not more than 0.1 inole of the complex per mole of enzyme can be isolatcd ( 6 1 ) .Of possible cxplanations for thesc findings, the following should bc considcrcd. First, the cnzy~ne priqmrutions rnny somctimrs contain a factor required for formation of the adcnylate complex, lack of this factor or its reinoval rcsulting in low yields. Second, cnaymcbound acetyl adenylate may bc in equilil)rium with an unstable enzymebound intermediate such that breakdown or dissociation of the latter results in low yields of the former; this liypothcsis would still 1)c consistent with a diralcnt cation bring rcquircrl for hinding of thc aclcnylatc 58. 1,. T. Wrhstcr, Jr., JBC 238, 4010 (1963). 59. A. H. Mehlrr nnd I<. Chttkrnburtty, A d ~ w u E . l ~ z y n t o l .35, 443 (1971). 60. L. T. Webster, Jr., JBC 242, 1232 (1967). 61. I,. T. Wrhstrr, Jr., unpuhlisllrd ohscrvntions.
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(see below). Finally, reccnt preparations of enzyme show a strong affinity for pyrophosphate and might contain this substrate (Section 111,B,5), thereby decreasing the yield of enzyme-bound adenylate. The significance of cnzyme-bound acctyl adenylatc will not he clear until these and other alternatives have 1)cen tested.
3. Cation Requirements
a. Divatent Metal Ions. With earlier preparations of ox heart enzyme two types of divalent metal ion requirements were demonstrated ( 6 2 ) . The first type, satisfied by millimolar concentrations of Mg2+,hln2+,Fez+, Co2+,or Ca2+,could be shown for the overall reaction and the first partial reaction, i t . , the formation of enzyme-bound acetyl adenylate from ATP, acetate, and enzyme [Eq. ( a ) ] Init not for the second partial reaction, namely, the formation of enzymc-hound acetyl adenylate from acetylCoA, AAIP, and enzyme [Eq. (1)) 1. Presumably, these divalent metal ions are required for chelation or charge neutralization of ATP and pyrophosphatc. An additional requirement for Ni", Cd", Fez+,or Cu2+ in concentrations approaching that of the enzyme was evident for the overall and both partial reactions ( 6 2 ) . A subsequent study with 63Ni showed that stoichiometric quantities of Ni2+and acetyl adenylate were bound to the enzyme and NiS+was required for adenylate binding (60). The proposcd requirement for cnzymc-bound divalent cation is difficult to reconcile with more recent results. Current preparations of enzyme, although catalytically more active, form w r y littlc enzymc-bound acetyl adcnylatc from substrates of the partial reactions (see above). Addition of NiCI, docs not increase the yield of cnzyme-bound acetyl adenylate and treatment of the enzyme and reaction components with Chelex, under conditions which formerly revealed a nickel requirement, does not significantly effect the overall reaction rate. Metal analysis of the enzyme revealed that it docs not contain significant quantities of divalent metal ions, although the small amount of co1)I)er found (0.12 mole/57,000 g of protein) exceeded the maximum yields of enzyme-hound adenylate formed from substrates (61, 6 3 ) . b. Monovalent Cations. With enzyme and reaction components prctreated to remove monovalent cations, an absolute dependence of activity on certain monovalent cations was shown. In the presence of N-2hydroxyrtl~ylpipcrazine-.~'-2-ethancsulfonicacid (HEPES) tctramethylammonium hydroxide I,uffer, pH 8, Na+ and Li+, previously recognized 62. L. T.Webster, Jr., J13C 240, 4164 (1965). 63. M.C. Scrritton, unpuhlishrd obs~rvntions.
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JOHN C. LONDESBOROUGH A N D LESLIE T. WEBSTER, J R .
only as inhibitors, were shown to fully activate the enzyme at low concentrations (5-8 mM) and to inhibit the reaction only a t higher concentrations (> 10 mM) (6‘4). The well-known activators K+, Rb+, NH,’ ( 6 5 ) , and tris+ (55) do not inhibit a t high concentrations. The effects of activating monovalent cations depend quantitatively on the concentrations of other monovalent cations, acetate and Mg2’ in a complex manner. The yield of enzyme-bound acetyl adenylate from substrates was also dependent on the concentration of activating monovalent cations (64). Enzyme-bound acetyl adenylate formed in the absence of activating monovalent cations by direct combination of enzyme and acetyl adenylate did not react with CoA unless activating monovalent cations were added (60). Although the molecular weight of the ox heart enzyme was the same in 0.13 M K’, Na+,or TMA’ (9),small conformational changes would not have been detected. 4. Selective Modification of Amino Acid Residues
The enzymes from rabbit heart (10) and ox heart (9) contain 8.7 2 0.2 and 8.0 & 0.5 sulfhydryl residues per 57,000 g of protein, respectively. Although acetyl-CoA synthetase, in common with most amino and fatty acid activating enzymes, is readily inhibited by sulfhydryl reagents such as p-mercuribenzoate there is no critical evidence that sulfhydryl residues are directly involved in the catalysis. The formation of ATP from acetyl adenylate and PPi was unaffected by complete titration of the rabbit heart enzyme with p-mercuribenzoate a t 0” (10). Only 4 residues in the ox heart enzyme react readily a t 0 ” ,resulting in complete inhibition of the overall back reaction ( 9 ) . This inhibition is fully reversible by millimolar concentrations of thiol. Thc inhibited enzyme, however, was still able to bind all of the substrates which could be tested in this system (CoA could not be tested). Reaction of the remaining 4 residues had a large energy of activation ( + 32 kcal/mole) and was first order with regard to enzyme and zero order with regard to p-mercuribenzoate concentration. These results suggest that the rate limiting step of the reaction of the “second” 4 residues may bc a conformational change in the protein which could account for the complete and irreversible inhibition observed when all 8 residues had reacted. The inhibition accompanying modification of the “first” 4 residues in the beef enzyme may also have resulted from a change in the enzyme’s shape. However, the results do not rigorously exclude a role for enzyme sulfhydryl in the part of the reaction mechanism involving CoA. T.Webstel,, Jr., JBC 241, 5504 (1966). R. W. von Korff, JHC 203, 265 (1953).
64. L.
65.
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Photoinactivation of the rabbit enzyme in the presence of methylene blue has a similar pH dependence as the photooxidation of histidine (10). A small decrease in the histidine content of the enzyme occurred during photoinactivation. The suggestion that histidine plays a role in the catalysis is attractive since Jencks has shown that imidazole catalyzes acyl transfer from acetyl adenylate to CoA (66). 5. Estimates of Substrate Afinity
All substrates, with the possible exception of CoA, altered the rate of the p-mercuribenzoate-induced irreversible inactivation of high specific activity, ox heart acetyl-CoA synthetase (9). Some caution is needed in interpreting the results, since the protection observed would involve binding of substrates to modified enzyme, i.e., 4 sulfhydryl residues have already reacted with p-mercuribenzoate. Such binding may differ from that to native enzyme. Adenosine triphosphate protected only in the presence of Mg'+ ions. The concentration of ATP causing half-maximal protection ( K %ATP) decreased from 260 p M a t pH 8.3 to less than 30 pkf a t pH 7.0. The nature of the dependence of the protection on ATP concentration indicated that all ATP binding sites were not equal and independent. In the presence of excess acetate and MgCl,, protection by ATP occurred a t such low concentrations that the enzyme could be titrated with ATP. Under these conditions, 2 moles of ATP were bound a t a lower enzyme concentration and more than two a t a higher enzyme concentration. The latter results also suggest that there are different kinds of ATP binding sites, but other explanations cannot be excluded except by more direct methods of measuring ATP binding. Protection by AMP did not require MgCl, and KH AMP was about 2 mM, both at pH 8.3 and pH 7.0. K , acetyl-CoA was about 1 mM. The binding of pyrophosphate appeared to be the tightest observed; K1,was less than 3 pM a t pH 7.0. This is of some interest since, according to the Berg mechanism, pyrophosphate does not react until after CoA has dissociated in the back reaction from the E -acetyl-CoA. AMP complex. 6. Steady State Kinetics and Reaction Mechanism
The most detailed kinetic study of the acetate:CoA ligase (AMP) has been made by Farrar and Ploughman (49,60)with partially purified ox lieart enzyme (specific activity 12 pm,oles/mg/min) . Michaelis constants for ATP, acetate, and CoA of 0.71 mM, 0.65 mM, and 0.21 &, respectively, were obtained in 0.1 M tris C1 0.2 M KC1 a t pH 8.3 and 66. W. P. Jencks and J. Carriuolo, JBC 234, 1272 (1957).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
37", with MgCl, in 0.4 m M excess of ATP; under these conditions the proportion of CoA which is Mg-chelated is nearly constant below 0.5 m M total CoA. Here and below, kinetic results will be discussed in terms of the general initial rate equation for a three substrate reaction enjoying linear kinetics, written according to Dalziel (67):
with A, B, and C standing for the concentrations of ATP, fatty acid, and CoA, respectively. The Berg mechanism is characterized by the and Farrar and Ploughabsence of the last three terms, i.e., +,,,, man reported parallel families of double-reciprocal plots when ATP was varied a t several CoA concentrations and constant acetate, and when acetate was varied a t several CoA concentrations and constant ATP, indicating, respectively, that the terms (+,,c + +,,!JB) and (+,,+abc/A) both approach zero. A triple transfer enzyme substitution mcchanism is excluded by the converging family of plots obtained when ATP was varied a t several acetate concentrations and constant CoA, i.e., +,,I, is positive. The absence of (Pat and +bc excludes quaternary complex mechanisms with either a rapid equilibrium random order or completely compulsory order of addition of substrates ( 6 7 ) .This kinetic pattern is consistent with the Berg mechanism, but also with a partially compulsory order quaternary complex mechanism in which CoA adds first to the enzyme followed by random addition of ATP and acetate ( 6 7 ) . The latter mechanism, which seems quite attractive, requires that be a finite term, but no evidence bearing on the existence of this term is available since all the data were obtained in the presence of a fixed concentration of a t least one suhstrate. Like the enzyme from yeast, enzymes prepared from ox or rabbit heart mitochondria can catalyze formation of acetyl-CoA or ATP from free acetyl adenylate and CoA or PP,, respectively. These reactions are probably fast enough to account for the overall forward and reverse reactions, respectively, although to our knowledge no data concerning the relative rates have been published. In contrast, the synthesis of acetyl hydroxamate from ATP, acetate, and hydroxylamine by both yeast and bcef heart enzymes is accelerated more than 100-fold by the addition of CoA (46, 5 6 ) . Coenzyme A therefore appears to be necessary either for the efficient synthesis or use of the acetate derivative that reacts with hydroxy lamine. Ox heart acetyl-CoA synthetase catalyzes an acetate-potentiated
+
67. K. Dalziel, BJ 114, 547 (1969)
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ATP-3’PPi exchange with a maximum rate, a t pH 8.0, of about 1% of the overall forward reaction rate ( 5 0 ) .The rate of the exchange increases with the acetate concentration up to 1 m M acetate, above which concentration acetate inhibits the reaction in an uncompetitive manner. Farrar interprets thesc data as being consistent with a compulsory order, Berg-type mechanism with A T P as the first substrate. However, some highly purified preparations of ox heart enzyme catalyze an ATP-PPi exchange in the absence of added acetate (61). C. MEDIUMCHAINFATTYACID:COALIGASES(AMP) 1. Substrates and Inhibitors
The broad specitivity of Mahler’s enzyme from ox liver mitochondria, including C,-C,, fatty acids and a variety of aromatic acids, was reviewed by ,Jencks (1). It now appears that both o-methoxylbenzoate and anthranilate can serve as suhstrates for this enzyme although salicylate and p-aminosalicylate cannot ( 1 4 ) .In tris buffer, pH 8, 50 pA4 salicylate, salicylamide, and gentisate inhibit hexanoyl-CoA formation from 0.25 mM hexanoate by 65, 35, and 30%, respectively (68). Mahler’s enzyme can be distinguished from a new “salicy1ate”:CoA ligase (AMP) partially purified from the same source. The latter is active with hexanoate, benzoate, o-methoxybenzoate, and anthranilate as well as with salicylate and p-aminosalicylate (14). Apparent K , values with salicylate as substrate are 1.4 & for salicylate and 14 pLM for ATP (68). This enzyme is strongly stimulated by pyrophosphatase and is stabilized by the combination of salicylate and ATP-Mg ( 1 4 ) . The intermediate chain fatty acid:CoA ligase from ox heart mitochondria shows activity with C,-C, fatty acids including isobutyrate and crotonate but not 0-hydroxybutyrate and acetoacetate ( 1 7 ) .Neither cysteine nor glutathione could substitute for CoA, G T P did not replace ATP, and the slight activities with ITP and C T P may have resulted from contamination with ATP. Unlike Mahler’s enzyme, the ox heart enzyme was not stimulated by pyrophosphatase and was only 50% inhibited by 8 mM pyrophosphate. Apparent K , values were 1.5 mM for butyrate, 3 mM for ATP, and 0.85 mM for CoA. Activation of medium chain enzyme in rat heart by 3‘,5‘-cyclic AMP and some other adenine nucleotides has been suggested (69). 68. P. Killenberg and L. T. Webster, Jr., unpublished observations. 69. V. G. Erwin, A. D. Anderson, and G . J. Eide, J . Pharm. Sn’. So, 77 (1971).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, JR.
2. Formation of Butyryl Adenylate
Butyryl adenylate has been isolated from the trichloroacetic acid supernatant of reaction mixtures containing [“C] butyrate, ATP, MgCl,, and large amounts of Mahler’s enzyme (70). Pyrophosphatase increased the yield of butyryl adenylate from 1.5 pmole/100,000g of enzyme of specific activity 0.28 pmole/min/mg although it did not alter the yield of acetyl adenylate obtained from acetyl-CoA synthetase.
3. Steady State Kinetics and Reaction Mechanism Bar-Tana et al. have carried out a kinetic analysis of the activation of butyrate by their Fractions I and I1 obtained from “Fraction C” of Mahler et a2. (11) by the additional steps of gel filtration and chromatography on DEAE (16). Fraction I1 exhibited linear kinetics which fitted equation (3) for the special case where +ab is absent or very small (71). I n particular, at high concentrations of the third substrate, the slopes of double reciprocal plots with butyrate or A T P as variable substrates increased as the concentration of CoA was decreased, indicating the existence, respectively, of both (+bc + +abC/A) and (+ac + +abc/B). ‘No cnzyme substitution mechanism has an initial rate equation containing either +abc or both +bc and +,,C. The slopes of the double reciprocal plots with butyrate as variable substrate a t constant CoA concentration were the same (parallel lines) over a range of A T P concentrations sufficient to cause a 4-fold increase in the intercept. This shows the probable absence of +&b which excludes a rapid equilibrium random order mechanism and fixes CoA as the middle substrate in a compulsory order quaternary complex mechanism. Apparent Michaelis constants with Fraction I1 a t pH 8.0 (tris) and 37” were ATP, 0.7 m M ; butyrate, 10 mM; and CoA, 0.2 mM. Fraction I proved less amenable to this type of analysis (72). BarTana et al. felt that the results fit an enzyme substitution mechanism of the Berg type. However, because of the small ranges of substrate concentrations and the nonlinearity of some of the double reciprocal plots, the data do not convincingly demonstrate the absence of +bc and +,,be. Since it also seems that Fraction I may be an altered form of Fraction I1 (16),the present reviewers would suggest that a quatcrnary complex mechanism must be seriously considered for both fractions. Apparent Michaelis constants for this fraction were still higher than for Fraction 11: ATP, 6 mM; butyrate, 20 mM; and CoA, 0.4 mM. The high K , 70. L. T. Webster, Jr. and F. Campagnari, JBC 237, 1050 (1962). 71. J. Bar-Tana and G. Rose, BJ 109, 283 (1968). 72. J. Bar-Tans, and G. Rose,BJ 109, 275 (1968).
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values that both fractions exhibited for some of the substrates may indicate some damage to the enzyme. Differences between the partial reactions of Fractions I and I1 of BarTana et d . were also observed. Both fractions catalyze the conversion of butyryl adenylate and CoA to butyryl-CoA a t rates greater than the overall forward reaction. This finding is consistent with the Berg mechanism. With Fraction I but not I1 the dependence of the rate of this partial reaction on the CoA concentration is strongly sigmoidal under certain conditions although results were variable (compare Figs. 8 and 9, ref. 72). In addition, Fraction I catalyzes the synthesis of A T P from butyryl adenylate and PPi, but Fraction I1 only very weakly catalyzes this partial reaction. It is characteristic of all Berg-type mechanisms, in which PPi must dissociate from the enzyme immediately before CoA reacts in the forward reaction, that PPi should be a simple competitive inhibitor of CoA (73). However, Graham and Park ( 7 4 ) , studying the activation of octanoate by Mahler’s enzyme, found that 1 mM PPi caused a 5-fold increase in the intercept of CoA double reciprocal plots without affecting the slope. This uncompetitive inhibition pattern is convincing evidence against a simple Berg mechanism, although the mechanism proposed by the authors must be regarded as highly tentative in view of the fragmentary nature of their data.
D. LONGCHAINFATTY ACID:COALIGASE(AMP) 1. Substrates and Inhibitors Long chain fatty acyl-CoA synthetases of the type first described by Kornberg and Pricer (76) have been difficult to purify and characterize because of their lipid environment and the low aqueous solubility of their substrates. Clearly, the type and concentration of detergent often present in the assay mixtures and the physical state of the fatty acid substrates may be expected to effect greatly the kinetics and substrate specificity of these enzymes. The recent development of an accurate, sensitive assay method based on the insolubility of long chain acyl-CoA’s in diethyl ether allowed Marcel and Suzue to study the kinetics of long chain fatty acid activation by rat liver microsomes a t p H 7.4 a t substrate concentrations below the critical micellar concentrations of both the fatty acids and their acyl-CoA products (27).These investigators found similar low 73. W. W. Cleland, BBA 67, 173 (1963). 74. A. B. Graham and M. V. Park, BJ 111,267 (1969). 75. A. Kornberg and W. E. Pricer, Jr., JBC 204, 329 (1953).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
values for K , (1-2 @ for four I) unsaturated fatty acids having a double bond in the A9 position (palmitoleate, oleate, linoleate, and linolenate) with values of V,,,,, ranging from 9.1 to 18.6 pmoles/hr/mg a t 37". Each of these fatty acids acted as competitive inhibitors of the activation of the others (apparent Ki values of 1.2 &). Palmitate showed a similar pattern ( K , of 2.8 pM; V,,, of 14.3 pmoles/hr/mg; Ki of 1.2 pM and 2.4 pkf against linolenate and palmitoleate, respectively). The apparent K , for CoA was similar for the activation of palmitate (7.2 p M ) and oleate (9.4 pM). The authors concluded that saturated and unsaturated fatty acids were probably activated by the same enzyme. Bar-Tana et al. (B),using older assay methods with material purified from rat liver microsomes (Section I,B,3), found some variations of K,, and V,,, with variations of the chain length and degree of saturation of the fatty acid substrates. At p H 8.0, the specific activity of this preparation with palmitate was 15 pmoles/hr/mg a t 37" and apparent Michaelis constants were ATP, 4.65 m M ; CoA, 50 @; and fatty acids, 11 p.M (C12) and 42 & (Clo), Compared to Marcel and Suzue's intact microsomes, this purified preparation had large K , values for fatty acid substrates and a comparable V,,,,, with palmitate. Because of the differences in methodology, these discrepancies may be apparent rather than real, but they should be recalled when interpreting the investigations of Bar-Tana et al. described below. 2. Partial and Exchange Reactions With the solubilized and purified enzymc (28) described in Section I,B,3, Bar-Tana et al. found that the maximum velocities of both partial reactions (viz, ATP formation and palmitoyl-CoA formation, respectively, from palmitoyl adenylate) were only 1% of that of the overall forward reaction rate ( 7 6 ) . Preincuhation of this enzyme preparation with ATY and CoA did not increase the rate of A T P formation from palmitoyl adenylate, although with an earlier less purified preparation such reactivation of this partial reaction could be shown (77). These partial reactions are readily catalyzed by intact microsomes. It is not yet clear whether their loss during solubilization of palmitoyl-CoA synthetase activity represents a change in the fundamental properties of palmitoyl-CoA synthetase or merely a purification. Both microsomes and the more highly purified enzyme catalyzed the incorporation of 32PYiinto ATP in the absence of all other substrates a t rates of 65% and 6.676, respectively, of thc overall forward reaction. The 76. J. Bar-Tana, G. Rose, and B. Shapiro, BJ 129, 1101 (1972) 77. J. Bar-Tana and €3. Shapiro, BJ 93, 533 (1904).
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significance of this exchange reaction, which is markedly inhibited by 0.2 mM palmitate, is far from clear, especially because small amounts of endogenous fatty acids are likely to be present in preparations derived from microsomes. I n a recent kinetic analysis of the forward reaction of the purified enzyme, Bar-Tana e t al. found that the sums (&,c + +abe/A) and (& + +abc/B) both approached zero, while (+ab + +abc/C) was positive (78). The evidence for, and interpretation of this kinetic pattern, is analogous to that concerning Farrar and Ploughman’s work with acetyl-CoA synthetase (see Section III,B,6), i.e., it is consistent with Berg’s enzyme substitution mechanism but also with a partially compulsory order quaternary complex mechanism. However, interpretation in the present case is also complicated by the fact that palmitate was probably present in the reaction mixtures partly as micelles and mixed micelles containing Triton X-100. Pulse labeling experiments were performed (78) to try to define a “stable” enzyme-substrate intermediate that should occur in any enzyme substitution mechanism (79).However, the results did not fit any simple pattern, and it is doubtful in any case whether pulse labeling experiments distinguish between “stable” and “unstable” enzyme-substrate complexes.
E. CONCLUSIONS Berg’s mechanism was originally proposed for acetyl-CoA synthetase from yeast, but has been widely assumed to describe all fatty acid:CoA ligases (AMP). It contains two distinct and independent postulates: (1) enzyme-bound acetyl adenylate is an obligatory intermediate and (2) PPi must dissociate from the enzyme immediately before CoA reacts in the forward reaction. There is good evidence that several ligases can make and use acyl adenylates. Enzyme-bound acetyl adenylate and butyryl adenylate can be isolated from reaction mixtures containing the respective activating enzymes. Yeast and heart acetyl-CoA synthetases, and possibly the microsomal long chain enzyme, catalyze fatty acid-dependent, CoAindependent [“PPi 1ATP exchanges. The acetyl-CoA synthetases, the medium chain enzyme of liver mitochondria, and some, but not all, preparations of microsomal long chain enzyme utilize acyl adenylates in partial reactions. However, these observations do not show that the acyl adenylates are obligatory intermediates. For all three enzymes, these 78. J. Bar-Tana, G. Rose, R. Brandes, and B. Shapiro, BJ 131, 199 (1973). 79. W.W.Cleland, BBA 67, 104 (1963).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
and other properties depend on the method of preparation. Thus, the recent high specific activity acetyl-CoA synthetase (Section I,B,l) can make only very small amounts of enzyme-bound acetyl adenylate, the medium chain enzyme can be resolved into varying proportions of two fractions, only one of which efficiently catalyzes the conversion of butyryl adenylate to ATP, and the long chain enzyme loses all partial reactions involving acyl adenylate during its purification. It is even possible that these partial reactions and the formation of enzyme-bound acyl adenylates are not inherent properties of the native enzymes. The second postulate of Berg’s mechanism, namely, that PPi must dissociate immediately before CoA binds, leads to kinetic predictions which are, in principle, easy to test. The reciprocal initial rate equation should contain no terms with both CoA and another substrate in the denominator, i.e., +8c = +bc = +abc = 0. The product inhibition pattern is also useful to identify this mechanism. Unfortunately, partly because of the instability of the various enzymes but mainly because there is no simple accurate assay procedure (see Section III,A), kinetic data of sufficient quality to show the absence of initial rate parameters are difficult to obtain. So far no study has been made in which low concentrations of all three substrates have been used in an attempt to test the existence of +abc. For ox heart acetyl-CoA synthetase, and possibly microsomal palmitoyl-CoA synthetase from rat liver, the kinetic data are consistent with Berg’s mechanism, but also with a partially ordered quaternary complex mechanism with CoA as the leading substrate and ATP and fatty acid subsequently adding randomly. For the medium chain enzyme, inhibition by PPi is not of the simple competitive type as required by the Berg mechanism. Furthermore, intersecting patterns of double reciprocal plots result when the concentrations of either ATP or butyrate are varied a t several CoA concentrations. These observations show that for certain preparations of medium chain acyl-CoA synthetase, PPi does not dissociate immediately before addition of CoA (Section IXI,C,3). The long chain enzyme solubilized from rat liver seems to require bound CoA for the expression of any activity (Section III,D,2). It therefore appears that dissociation of PPi may not precede reaction with CoA and therefore a quaternary eneyme-substrate complex occurs. This opens up the possibility for concerted mechanisms, although the transitory occurrence of acyl adenylates in the central enzyme-substrate complexes cannot be excluded by initial rate data. A satisfactory mechanism has not been convincingly established for any fatty acid:CoA ligase (AMP), and the information presently available suggests there may be important mechanistic differences between the members of this class of enzymes.
LESLIE T. WEBSTER, JR.
I. Introduction . . . . . . . . . . . A. Scope of This Chapter . . . . . . . B. Distribution and Isolation of Acid:CoA Ligases . 11. Molecular Properties of Acetate:CoA Ligase (AMP) . . 111. Catalytic Properties of the Fatty Acid:CoA Ligases (AMP) A. General Considerations . . . . . . . B. Acetate:CoA Ligase (AMP) . . . . . . C. Medium Chain Fatty Acid:CoA Ligases (AMP) , D. Long Chain Fatty Acid:CoA Ligase (AMP) . . E. Conclusions . . . . . . . . . .
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469 469 470 474 475 475 477 483 485 487
I. Introduction
A. SCOPE OF THIS CHAPTER The fatty acyl-CoA synthetases include the ATP-dependent fatty acid :CoA ligases (AMP) and the GTP-dependent fatty acid :CoA ligases (GDP), which catalyze reactions ( 1 ) and (2), respectively. Me¶+
+ ATP’- + COASH RCO*SCoA+ AMPa- + PP;’Met+ RCO.SCoA + GDPS- + PipRCOO- + GTP4- + COASH RCOO-
(1) (2)
The reader is referred to Jenck’s previous review of fatty acid activation ( 1 ) . In the present chapter, emphasis is placed on more recent work, particularly that concerning the short, medium, and long chain fatty 1. W. P. Jencks, “The Enzymes,” 2nd ed., Vol. 6, p. 373, 1962. 469
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER,
JR.
acid: CoA ligases (AMP). Information about other acyl-CoA synthetases, including the medium-long chain fatty acid:CoA ligase (GDP) of Rossi and co-workers ( 2 ) is generally much less complete but is summarized, together with distribution data and isolation methods, in Section 1,B. Rossi and Gibson’s enzyme is mechanistically related to the succinate: CoA ligases (ADP and GDP) which are treated in Chapter 18 (3).
B. DISTRIBUTION AND ISOLATION OF ACID:CoA LIGASES 1. Acetate:CoA Ligase ( A M P )
The acetate:CoA Ligase (AMP) (EC 6.2.1.1) enzyme has been purified 20-30-fold from several new sources including ox brain, Euglena gracilis, and Aspergillus niger (4-6) ; i t remains to be demonstrated in heterotrophic bacteria. Acetyl-CoA synthetase probably has a bimodal distribution in mammalian cells, being found in both mitochondria1 and supernatant fractions of rat liver, kidney, heart, and adipose tissue ( 7 ) . The most highly purified enzyme has been obtained from ox heart mitochondria by two different methods. Unstable, aggregating, but niicrocrystalline and apparently homogeneous material with a specific activity of 35 pmoles/min/mg a t 37” was prepared earlier by Webster (8).Recently, more stable but noncrystalline material of higher specific activity (66 pmoles/min/mg) was obtained by a modification in which a pH fractionation and neomycin step was substituted for negative absorption with alumina Cy gel (9). Sharkova has purified acetyl-CoA synthetase from rabbit heart by the earlier method to a specific activity of 12 pmoles/min/mg at 37” (10). 2. Medium Chain Fatty A c i d : C o A Ligases ( A M P ) The medium chain fatty acid:CoA ligasc (AMP) (EC 6.2.1.2) activity was first partially purified from beef liver mitochondria by Mahler et al. 2. L. Galzigna, C. R. Rossi, L. Sartorelli, and D. M. Gibson, JBC 242, 2111 (1967). 3. W. Bridger, Chapter 18, this volumc. 4. G. A. Rao, I. A. Hansen, and B. K. Bnchhawat, J . Sci. Znd. Res. Sect. C 20, 284 (1961). 5 . E. Ohrnann, BBA 82, 325 (1964). 6. V. K. Shah and C. V. Ramakrislinan, Euzymologin 26, 53 (1963). 7. C. Barth, M. Sladek, and K. Decker, BRA 248, 24 (1971). 8. L. T. Webster, Jr., “Methods in Enzymology,” Vol. 13, p. 375, 1969. 9. J. C. Londesborough, S. L. Yuan, and L. T. Webster, Jr., BJ 133, 23 (1973). 10. E. V. Sharkova, Biokhimiya 33, 792 (1968); Biochemistry ( U S S R ) 33, 648 (1968).
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(11) and since reported in a large variety of mammalian tissues and higher plants ( 1 ) . Newer sources include yeast and pseudomonads (19, IS). Mahler’s fraction was active with a variety of aliphatic and aromatic carboxylic acids (Section III,C,l) . Several different medium chain fatty acyl-CoA synthetases may occur in the same subfraction of a mammalian tissue. The hexanoate:CoA ligase (AMP) activity of beef liver mitochondria has been resolved by Killenberg et al. (14) into two fractions, both of which can activate benzoate but only one of which, purified 30fold, can activate salicylate or p-aminosalicylate. This source also contains the medium-long chain fatty acid:CoA ligase (GDP) ( 8 ) . Bar-Tana et al. have resolved the butyrate:CoA ligase (AMP) activity from Mahler’s “Fraction C” into two subfractions which may, however, be derived from a single protein (15).Again, crude extracts of dog kidney mitochondria have both butyryl-CoA and benzoyl-CoA synthetase activities, but a fraction which activates butyrate, but not benzoate, can be obtained from them (16). The substrate specificity of medium chain fatty acid:CoA ligases (AMP) may also vary with the tissue of origin. Thus, a n enzyme has been purified from beef heart mitochondria to a specific activity of 3.3 pmoles butyryl-CoA/min/mg a t 37” which, unlike related enzymes in beef liver mitochondria (11, 1 4 ) , does not activate aromatic fatty acids or aliphatic acids with more than seven carbon atoms (17).To date, no acid :CoA ligases (AMP) have been isolated which exclusively activate aromatic carboxylic acids. Plants may contain such enzymes [e.g., the CoA ligase (AMP) activity observed with cinnamic acids in extracts of spinach leaves, peas, runner beans, and parsley cell cultures], but their substrate specificities remain to be determined (18, 19).
3. Long Chain Fatty Acid:CoA Ligase ( A M P )
The long chain fatty acid:CoA ligase (AMP) (EC 6.2.1.3) type of activity has been found in particulate subfractions of many mammalian 11. H. It. Mahlcr, S. J. Wakil, and R. M. Bock, JBG 204, 453 (1953). 12. T. J. Trust and N. F. Millis, J. Bucten’ol. 104, 1397 (1970). 13. T. J. Trust and N. F. Millis, J. Bacterial. 105, 1216 (1971). 14. P. G. Killcmberg, E. D. Davidson, and L. T. Webster, Jr., Mol. Pharm. 7, 260 (1971). 15. J. Bar-Tana, G. Rosc, and B. Shapiro, BJ 109, 269 (1968). 16. L. T. Webster, Jr. and Z. R. Vlahcevic, unpublished observations. 17. I,. T. Wcbster, Jr., L. D. Gerowin, and L. Rakita, JBC 240, 29 (1965). 18. E. Walton and V. S. Butt, Phytochemktry 10, 295 (1971). 19. K. Hahlbrock and H. Griseback, FEBS Lelt. 11, 62 (1970).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, JR.
tissues (see, e.g., refs. 20 and 21) and has been reported in pseudomonads (IS), yeast ( 2 2 ) , and Bacillus megaterium (23); the activity is inducible in Escherichia coli (24, 26). The microsomal activities arc difficult to solubilize ; the microsomal enzyme of hamster intestinal mucosa may even occur as part of a glyceride synthetase complex containing a t least four different polypeptide chains ( 2 6 ) .Marcel and Suzue (27) have presented kinetic evidence that a single enzyme activates most, if not all, saturated and unsaturated long chain fatty acids in rat liver microsomes (Section II1,D). Bar-Tana et al. have solubilized palmitoyl-CoA synthetase in apparently quantitative yield from rat liver microsomes by treatment with sodium deoxycholate after their extraction with dry organic solvents (28). The extracted material was purified another 14-fold to a specific activity of 0.25 pmole/min/mg a t 37". Specific activity was constant throughout a single protein peak observed by gel filtration; a molecular weight of 250,000 was estimated under conditions where detergent was not completely removed. The activity of this preparation was increased by a great variety of detergents, although these had little effect on intact microsomes. In contrast, the soluble long chain acyl-CoA synthetase partially purified by Massaro and Lennarz from the 78,000-g supernatant of B . megaterium was not activated by any of many detergents tested and was severely inhibited by somc (29).The last enzyme requires ATP, but it has not been shown whether the nucleotide product is AMP or ADP. 4. Medium-Long Chain Fatty A r i d : P o A Ligase ( G D P ) Rossi and co-workers have obtained two partially purified preparations with GTP-dependent fatty acyl-CoA synthctase activity from rat liver mitochondria. Activity wit11 C,-C,, fatty acids was purified from acetonctreated mitochondria ( X I ) , whereas a preparation equally active with 20. S. V. Pande and J. F. Mead, BRA 152, 636 (1968). 21. K. Lippel and D. S. Henttir, BBA 218, 227 (1970). 22. %. Duvnjnk, J. M. Lebeault, B. Rorlie, and E. Azoulny, BBA 202, 447 (1970). 23. W. J. Lennarz, BBA 73, 335 (1963). 24. G. Wrrks, M. Shapiro, li. 0. Burns, and S. J. Wakil, J . Bncleriol. 97, 827 ( 1969). 25. P. Ovcrsth, G . Pnuli, nnd 11. IJ. Srhnirrr, Rur. J. Biochem. 7, 559 (1969). 26. G. A. Rao and J. M. Jolinston, BBA 125, 465 (1966). 27. Y. L. Marcel and G. Suzuc, JBC 247, 4433 (1972). 28. J. Bar-Tann, G. Rose, nnd n. Stinpiro, BJ 122, 353 (1972). 29. E. J. Massaro and W. J. Lennarz, Biochemistry 4, 85 (1965). 30. C. R. Rossi and D. M. Gibson, JUG' 239, 1694 (1964).
14.
FATTY
ACYL-COA SYNTHETASES
473
both butyrate and oleate (0.1 pmoleJmin/mg a t 38”) was obtained from mitochondria not exposed to acetone ( 8 ) . Both preparations were free from succinyl-CoA synthetase and ATP-dependent acyl-CoA synthetases. Extraction with acetone of the preparation active with long chain substrates removed lipid and caused a 70% loss of both butyryl-Coh and oleoyl-CoA synthetase activities ( 3 1 ) . However, acetone extraction of sonicated mitochondria decreased only the long chain acyl-CoA synthetase but not the butyryl-CoA synthetase activity. I n both cases, addition of lecithin largely restored the activities lost during acetone treatment. The authors suggested that the different specificity of the isolated GTP-dependent enzymes for fatty acid substrates results from their different lecithin contents, but it is not yet clear if the enzymes are identical in other respects. The long chain enzyme was estimated to have a molecular weight of 20,000 by gel filtration ( 2 ) . Michaelis constants were oleate, 2.1 mM; palmitate, 3.3 mM; butyrate, 0.22 m M ; octanoate, 0.2 mM; CoA, 3.3 m M ; and GTP, 4.3 mM. The enzyme was completely inhibited by 2 mM F- and 70% inhibited by 5 mM phosphate, whereas these reagents do not inhibit long chain fatty acid:CoA ligases (AMP). Rossi et al. have now shown that the GTP-dependent enzyme contains 1 mole of 4’-phosphopantetheine/2O1000g, which is essential for activity (32). In this respect also, the enzyme resembles succinate:CoA ligase (GDP) ( 3 ) ,and differs from the fatty acid:CoA ligases (AMP) in which such a cofactor has not been demonstrated. 5. Other Related Acid:CoA Ligases
A distinct oxalic acid:CoA ligase (AMP) occurs in seeds of Lathyrus sativa (33), and lactyl-CoA synthetase activity has been found in E . coli ( 3 4 ) . A specific propionyl-CoA synthetase has been purified from sheep liver ( 3 5 ) . Acetoacetyl-CoA synthetase activity, first reported in pigcon liver by Stern and Ochoa ( 3 6 ) , is present in a wide variety of tissues including rat liver (3’7). An analogous, but GTP-linked enzyme occurs in mitochondria from brown adipose tissue and requires 4’-phos31. I,. S:irtordli, I,. Galzigna, C. R. Rossi, and D. M. Gibson, BBRC 26, 90 (1967). 32. C. R. Rossi, A. Alexandre, L. Galzigna, L. Sartorelli, and D. M. Gibson, JBC 245, 3110 (1970). 33. G. A. R. Johnston and H. J. Lloyd, Aust. J . Biol. Sei. 20, 1241 (1967). 34. R. E. Mcgraw, H. C. Reeves, and S. J. Ajl, J . Bacterial. So, 984 (1965). 35. S. B. Latimer, Ph.D. Dissertation, Univeraity of North Carolina, Raleigh, 1967. 36. J. R. Stcrn and S. Ochoa, JBC 191, 161 (1951). 37. J. R. Stern, BBRC 44, 1001 (1971).
474
JOHN C. LONDESBOROUGH AND LESLIE T. WBSTER, JR.
phopantetheine (38).A possibly specific cholic acid: CoA ligase (AMP) is present in the microsomes of guinea pig and rat (39). The glutarylCoA synthetase from pigeon liver and dog muscle is cqually active with ATP, GTP, and ITP ; nucleotide diphosphate and inorganic phosphate are formed (40). Glutaryl-CoA synthctase activity is also found in Pseudomonas fluorescens and Rhodopseudomoms spheroides (41, @) .
It. Molecular Properties of Acetate :CoA Ligase (AMP)
Few of the enzymes listed in Section I,B have been purified sufficiently to permit studies of their molecular propcrtics and only acetyl-CoA synthetase from heart mitochondria has been examined in any detail. Gel filtration experiments conducted with protein concentrations over a 1000-fold range indicated a constant molecular weight of 57,000k 3500 for the high specific activity preparations of acetyl-CoA synthetase from ox heart mitochondria (9). With the essentially homogeneous but unstable enzyme prepared earlier, molecular weights estimated by sedimentation equilibrium ranged from 30,000 a t the meniscus to greater than 100,OOO a t the base, and depended on the particular preparation and its age (4.9).Two peptides in the 27,000to 30,000molecular weight region can be demonstrated by sodium dodecyl sulfate (SDS) disc gel electrophoresis after the protein is oxidized with performic acid, whereas a broad band appears in this region after the protein is reduced in SDSmercaptoethanol (4).Whether such peptides in fact represent nonidentical subunits of the native protein as described for tryptophanyltRNA synthetase from beef pancreas (45) remains to be established. The amino acid analysis of the highest specific activity preparations was unremarkable and accounted for 93.5% of the dry weight (9). No p-alanine, amino sugars, neutral sugars, or sialic acids could be demonstrated. By dry weight analysis the extinction coefficient a t 280 nm is 1.41 mg-’ cm2. The ultraviolet spectrum displays the usual peak a t 280 38. C. R. Roe& Z. Drahota, A. Alexandre, and N. Siliprandi, Abstr. FEBS Meet., 6th, 1969 p. 84 (1969). 39. W. H. Elliott, “Methods in Enzymology,” Vol. 5, p. 473, 1962. 40. G. K. K. Menon, D. L. Friedman, and J. R. Stern, BBA 44, 375 (19eo). 41. Y . Nishizuka, S. Kuno, and 0. Hapaishi, BBA 43, 357 (1960). 42. S. Lartillot, J. Dedreux, and C. Baron, Bull. SOC.Chim. B i d . 47, 919 (1965). 43. L. T. Webster, Jr., JBC 2a0, 415 (1965). 44. L. T. Webster, Jr. and L. Aldwin, unpublished observations (1973). 45. E. C. Preddie, JBC 244, 3958 (1969).
14.
FATTY
475
ACYL-COA SYNTHETASES
nm and a rather high 280/260 absorption ratio (1.9-2.0 a t pH 8 ) . The absorptions a t 294.4 nm and 280 nm in 0.1M KOH are entirely consistent with the tyrosine and tryptophan contents. Fully active preparations do not therefore appear to contain significant amounts of nucleotides or pantothenate derivatives. The rabbit heart enzyme appears homogeneous in the ultracentrifuge with a sedimentation coefficient of 3.86 (10) as compared to values of s20,wranging from 3.5 to 4.8 for different preparations of the ox heart enzyme (9, 43). 111. Catalytic Properties of the Fatty Acid:CoA Ligares (AMP)
A. GENERAL CONSIDERATIONS In addition to the overall reaction, many fatty acid:CoA ligases (AMP) catalyze several partial reactions. The synthesis of hydroxamates from fatty acids, ATP, and hydroxylamine, and the synthesis of ATP from acyl adenylate and PPi are Mg-dependent but CoA-independent. The synthesis of acyl-CoA’s from acyl adenylates and CoA is Mgindependent. Berg therefore proposed that the overall reaction proceeds via an acyl adenylate intermediate (46) : M#+
+ RCOOH + E E.RCO.AMP + PPi E.RCO*AMP+ COA+ E + RCO-COA + AMP ATP
(a)
(b) This mechanism was supported by Boyer’s finding of an IRO exchange between fatty acid and AMP as well as by other exchange reactions discussed by Jencks (1). It has since been established that enzyme-bound acetyl adenylate and butyryl adenylate can be made from ATP, acetate, or butyrate and large amounts of the respective activating enzyme under appropriate conditions (Sections II1,B and C) . However, while acyl adenylates are good substrates for most, if not all, fatty acid:CoA ligases, it is far from clear whether they are obligatory intermediates in the overall reaction. Particular attention will be paid to this point in the remainder of the review. Investigations of the mechanisms of the fatty acyl-CoA synthetases are still hindered by unsatisfactory assay procedures. Acetyl-CoA synthetase activity can be monitored by coupling the reaction with those of citrate synthase and malate dehydrogenase, thereby allowing the con46. P. Berg, JBC 222, 991 (1956).
476
JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
tinuous measurement of NADH production spectrophotometrically (47, 48). However, components of the coupled system have been reported to interfere with acetyl-CoA synthetase activity (49, 5 0 ) . The sensitivity of methods using the thioester absorption a t 232 nm (49, 50) is limited by the high blank absorption a t even moderate CoA and A T P concentrations. Assays for free CoA thiol or acyl-CoA (c.g., refs. 15 and 28) have been discontinuous except recently (see ref. 7 8 ) and suffer from the usual drawbacks of poor sensitivity and accuracy. However, thc differential extraction of radioactively labeled free acids from acyl-CoA (e.g., refs. 14 and 2 7 ) , while laborious, ran give data of high quality with the more lipophilic fatty acids. Under the conditions usually used to assay thesc enzymes (pH > 7, excess Mg2+ions) more than 95% of the total A T P is present as MgATY2which is probably the true substrate. It is not known whether the enzymes can distinguish between the uncomplexed and metal chelate forms of CoA and its esters. Little information about the strength of these complexes is available. The shift of the titration curves of 8hydroxyquinoline with MgCI, in the presence and absence of CoA ran be accounted for by the formation of MgCoA with an apparent stability constant of 250 M-l in 0.24 M tris CI, p H 7.7 a t 25" ( 5 1 ) .Approximately 50% of the total CoA is therefore chelated a t a free Mgz+ concentration of 4 mM. The interpretation of kinetic data for the fatty acid activating enzymes is complicated by these additional equilibria. By comparison of the equilibria of the citrate lyase and citrate synthase reactions a t pH 7.2, 25", and p. = 0.1 M , Tate and Datta ( 5 2 ) obtained a value of -6.8 kcal/mole for the free energy of hydrolysis (AG',) of acetyl-CoA to CoA and acetate anion. This is in good agreement with the value of -7.7 kcal/mole which Jencks et d.estimated from a study of the isomerization of mercaptopropyl acetate a t pH 7.0, 39", and p = 0.3M ( 5 3 ) . All the acyl-CoA synthetase reactions are therefore expected to be freely reversible. For acetyl-CoA synthetase and the medium chain enzyme equilibrium constants close to unity have been observed (11, 47, 5 4 ) , but the effects of temperature, ionic strength, and 47. P. Hcle, JBC 206, 671 (1954). 48. D. J. Pearson, BJ 95, 23c (1965). 49. W. W. Farrar and F. M. Ploughman, Fed. Proc., Fed. Amer. SOC.Ezp. Biol.
29, 425 (1970). 50. W. W. Farrar, personal communication. 51. J. C. Londesborough, unpublished observations. 52. S. S. Tate and S. P. Datta, BJ 94, 470 (1965). 53. W. P. Jencks, S. Cordes, and J. Carriuolo, JBC 235, 3608 (1960). 54. M. E. Jones, Fed. Proc., Fed. Amer. SOC. Exp. Biol. 12, 708 (1953).
14.
FATTY
477
ACYL-COA SYNTHETASES
metal ion concentration have not been studied. This information is eventually required for comparison with kinetic data. B. ACETATE :CoA LIGASE (AMP) 1. Substrates and Inhibitors Besides acetate, propionate, and acrylate ( 4 7 , 5 5 ) ,the ox heart enzyme activates isobutyrate, fluoroacetate, and probably formate (66). Rates for the last three substrates are far lower than that for acetate. Evidence that formate is a substrate is indirect; it supports a CoA-dependent breakdown of ATP. Net thiol disappearance was not shown, presumably because formyl-CoA was unstable under the assay conditions ehployed. The ox heart enzyme appears quite specific for adenine nucleotides ( 5 6 ) . At 3.6 mM, dATP gives approximately 70% the rate obtained with ATP. Neither glutathione nor pantetheine can substitute for CoA as the acyl acceptor. However, with the yeast preparation, Gunther and Mautner have shown that seleno-coenzyme A can act as both a substrate and a competitive partial inhibitor of coenzyme A ( 5 7 ) . At sufficiently high concentrations, many fatty acid substrates inhibit the rate of acyl-CoA formation (55). Acetyl-CoA synthetase may also be inhibited by certain buffers. We have recently found that the decrease in rate in tris buffer below pH 8 does not occur in piperazine-l\r,ZV’bis [ethanesulfonic acid] (PIPES) buffer and cannot be explained by changes in Michaelis constants (Table I). The inhibition by tris a t TABLE I OF BUFFER AND pH ON THE MICHAELIS CONSTANTS AND TURNOVER EFFECT NUMBER IN THE FORWARD REACTION FOR ACETYL-COA SYNTAETASE FROM Ox HEART MITOC~ONDRIA~ ~~
Parameter
TN (moles/min/ 270 (230 at 57,000 g enzyme) 20 mM K+) 0.35 K , ATP (mM) 0.22 K,,, acetate (mM) 0.22 K , CoA (mM) (I
~~
Tris, pH 8.3 PIPES, pH 7.8 (200 mM K+) (20 mM K+) 230
-
PIPEX3, pH 6.7 TI%, pH 6.7 (200 m M K+) (200 mM K+) 280
0.10 0.14 1.2
32 0.017 c2.0 0.28
30”, 5 mM MgC12, 100 mM tris or 75 m M PIPES.
55. F. Campagnari and L. T. Webster, Jr., JBC 238, 1628 (1963). 56. J. C. Londesborough and L. T. Webster, Jr., unpublished observations. 57. W. H. H. Gunther and H. G . Mautner, unpublished observations cited by H. G. Mautner, “Methods in Enzymology,” Vol. 18A, p. 338,1970.
478
JOHN C. LONDESBOROUGH AND LESLIE T. W'EBSTER, J R .
neutral pH is not undcrstood. PossMy the interaction hctwccn enzyme and tris cation, which at higher pH activates the cnzymc in an analogous fashion to XH,' and K', changcs to tlic inhibitory pattern characteristic of Na' (Section III,B,3). Thr pA of the change in maximum wloeity in tris is about 7.3; tliiis, an ionization of the enzyme, ratlicr than the tris (pk' 8.3 a t 30°),appears rcsponsiblc. 2. F o r m t i o n of Enzynie-Bound Acetyl Adenylnte
As discusscd prcviously by .Jencks ( 1 ) initial failures to dcmoiistrate enzymic formation of ncctyl adcnylatc constituted a major drawl)ack to Berg's proposcd mechanism. This objcction was later remorrd whcn Wehster rcportcd tlic isolation of cnzymc-bound acctyl adcmylatc by gel filtration of rcaction mixturcs containing [l'C] acctatc, ATP-lIg, and substratc quantities of highly purified acctyl-CoA synthetasc from ox heart (58). As cxpcctcd, thc cnzyme-1)ound species was also synthcsizcd from acetyl-CoA and AMP arid rcactcd with CoA to form acetyl-CoA. This was the first observation of an isolatcd cnzyinc-hound spccics for any of thc twid:acccptor ligascs (AJIP) , and thc sanic approach was subsequently utilizcd to isolate cnzymc-bound aminoacyl adcnylatcs from a variety of amino acid-tRSA synthctasr systems (59). Howcvcr, whcthcr cnzymc-hound acrtyl adcnylatr is an obligatory intermediate in thc acetyl-CoA synthctasc rcaction is unccrtain, and yields of this complex from suhstratrs of thc partial reactions have varied markcdly dcpcnding on tlic preparation of cnzymc. Higher yicltls wcrc obtained earlirr with prcparations of variahlc stability and purity (43, 58, 60) ; the highest was found with a partially purificd cnzyiiic which had not been subjcctcd to gcl filtration during purification (60).\\'it11 the recent enzyme preparations, which are more stable and catalytically active, not more than 0.1 inole of the complex per mole of enzyme can be isolatcd ( 6 1 ) .Of possible cxplanations for thesc findings, the following should bc considcrcd. First, the cnzy~ne priqmrutions rnny somctimrs contain a factor required for formation of the adcnylate complex, lack of this factor or its reinoval rcsulting in low yields. Second, cnaymcbound acetyl adenylate may bc in equilil)rium with an unstable enzymebound intermediate such that breakdown or dissociation of the latter results in low yields of the former; this liypothcsis would still 1)c consistent with a diralcnt cation bring rcquircrl for hinding of thc aclcnylatc 58. 1,. T. Wrhstcr, Jr., JBC 238, 4010 (1963). 59. A. H. Mehlrr nnd I<. Chttkrnburtty, A d ~ w u E . l ~ z y n t o l .35, 443 (1971). 60. L. T. Webster, Jr., JBC 242, 1232 (1967). 61. I,. T. Wrhstrr, Jr., unpuhlisllrd ohscrvntions.
14.
FATTY
ACYL-COA STNTHETASES
479
(see below). Finally, reccnt preparations of enzyme show a strong affinity for pyrophosphate and might contain this substrate (Section 111,B,5), thereby decreasing the yield of enzyme-bound adenylate. The significance of cnzyme-bound acctyl adenylatc will not he clear until these and other alternatives have 1)cen tested.
3. Cation Requirements
a. Divatent Metal Ions. With earlier preparations of ox heart enzyme two types of divalent metal ion requirements were demonstrated ( 6 2 ) . The first type, satisfied by millimolar concentrations of Mg2+,hln2+,Fez+, Co2+,or Ca2+,could be shown for the overall reaction and the first partial reaction, i t . , the formation of enzyme-bound acetyl adenylate from ATP, acetate, and enzyme [Eq. ( a ) ] Init not for the second partial reaction, namely, the formation of enzymc-hound acetyl adenylate from acetylCoA, AAIP, and enzyme [Eq. (1)) 1. Presumably, these divalent metal ions are required for chelation or charge neutralization of ATP and pyrophosphatc. An additional requirement for Ni", Cd", Fez+,or Cu2+ in concentrations approaching that of the enzyme was evident for the overall and both partial reactions ( 6 2 ) . A subsequent study with 63Ni showed that stoichiometric quantities of Ni2+and acetyl adenylate were bound to the enzyme and NiS+was required for adenylate binding (60). The proposcd requirement for cnzymc-bound divalent cation is difficult to reconcile with more recent results. Current preparations of enzyme, although catalytically more active, form w r y littlc enzymc-bound acetyl adcnylatc from substrates of the partial reactions (see above). Addition of NiCI, docs not increase the yield of cnzyme-bound acetyl adenylate and treatment of the enzyme and reaction components with Chelex, under conditions which formerly revealed a nickel requirement, does not significantly effect the overall reaction rate. Metal analysis of the enzyme revealed that it docs not contain significant quantities of divalent metal ions, although the small amount of co1)I)er found (0.12 mole/57,000 g of protein) exceeded the maximum yields of enzyme-hound adenylate formed from substrates (61, 6 3 ) . b. Monovalent Cations. With enzyme and reaction components prctreated to remove monovalent cations, an absolute dependence of activity on certain monovalent cations was shown. In the presence of N-2hydroxyrtl~ylpipcrazine-.~'-2-ethancsulfonicacid (HEPES) tctramethylammonium hydroxide I,uffer, pH 8, Na+ and Li+, previously recognized 62. L. T.Webster, Jr., J13C 240, 4164 (1965). 63. M.C. Scrritton, unpuhlishrd obs~rvntions.
480
JOHN C. LONDESBOROUGH A N D LESLIE T. WEBSTER, J R .
only as inhibitors, were shown to fully activate the enzyme at low concentrations (5-8 mM) and to inhibit the reaction only a t higher concentrations (> 10 mM) (6‘4). The well-known activators K+, Rb+, NH,’ ( 6 5 ) , and tris+ (55) do not inhibit a t high concentrations. The effects of activating monovalent cations depend quantitatively on the concentrations of other monovalent cations, acetate and Mg2’ in a complex manner. The yield of enzyme-bound acetyl adenylate from substrates was also dependent on the concentration of activating monovalent cations (64). Enzyme-bound acetyl adenylate formed in the absence of activating monovalent cations by direct combination of enzyme and acetyl adenylate did not react with CoA unless activating monovalent cations were added (60). Although the molecular weight of the ox heart enzyme was the same in 0.13 M K’, Na+,or TMA’ (9),small conformational changes would not have been detected. 4. Selective Modification of Amino Acid Residues
The enzymes from rabbit heart (10) and ox heart (9) contain 8.7 2 0.2 and 8.0 & 0.5 sulfhydryl residues per 57,000 g of protein, respectively. Although acetyl-CoA synthetase, in common with most amino and fatty acid activating enzymes, is readily inhibited by sulfhydryl reagents such as p-mercuribenzoate there is no critical evidence that sulfhydryl residues are directly involved in the catalysis. The formation of ATP from acetyl adenylate and PPi was unaffected by complete titration of the rabbit heart enzyme with p-mercuribenzoate a t 0” (10). Only 4 residues in the ox heart enzyme react readily a t 0 ” ,resulting in complete inhibition of the overall back reaction ( 9 ) . This inhibition is fully reversible by millimolar concentrations of thiol. Thc inhibited enzyme, however, was still able to bind all of the substrates which could be tested in this system (CoA could not be tested). Reaction of the remaining 4 residues had a large energy of activation ( + 32 kcal/mole) and was first order with regard to enzyme and zero order with regard to p-mercuribenzoate concentration. These results suggest that the rate limiting step of the reaction of the “second” 4 residues may bc a conformational change in the protein which could account for the complete and irreversible inhibition observed when all 8 residues had reacted. The inhibition accompanying modification of the “first” 4 residues in the beef enzyme may also have resulted from a change in the enzyme’s shape. However, the results do not rigorously exclude a role for enzyme sulfhydryl in the part of the reaction mechanism involving CoA. T.Webstel,, Jr., JBC 241, 5504 (1966). R. W. von Korff, JHC 203, 265 (1953).
64. L.
65.
14.
FATTY
ACYL-COASYNTHETASES
481
Photoinactivation of the rabbit enzyme in the presence of methylene blue has a similar pH dependence as the photooxidation of histidine (10). A small decrease in the histidine content of the enzyme occurred during photoinactivation. The suggestion that histidine plays a role in the catalysis is attractive since Jencks has shown that imidazole catalyzes acyl transfer from acetyl adenylate to CoA (66). 5. Estimates of Substrate Afinity
All substrates, with the possible exception of CoA, altered the rate of the p-mercuribenzoate-induced irreversible inactivation of high specific activity, ox heart acetyl-CoA synthetase (9). Some caution is needed in interpreting the results, since the protection observed would involve binding of substrates to modified enzyme, i.e., 4 sulfhydryl residues have already reacted with p-mercuribenzoate. Such binding may differ from that to native enzyme. Adenosine triphosphate protected only in the presence of Mg'+ ions. The concentration of ATP causing half-maximal protection ( K %ATP) decreased from 260 p M a t pH 8.3 to less than 30 pkf a t pH 7.0. The nature of the dependence of the protection on ATP concentration indicated that all ATP binding sites were not equal and independent. In the presence of excess acetate and MgCl,, protection by ATP occurred a t such low concentrations that the enzyme could be titrated with ATP. Under these conditions, 2 moles of ATP were bound a t a lower enzyme concentration and more than two a t a higher enzyme concentration. The latter results also suggest that there are different kinds of ATP binding sites, but other explanations cannot be excluded except by more direct methods of measuring ATP binding. Protection by AMP did not require MgCl, and KH AMP was about 2 mM, both at pH 8.3 and pH 7.0. K , acetyl-CoA was about 1 mM. The binding of pyrophosphate appeared to be the tightest observed; K1,was less than 3 pM a t pH 7.0. This is of some interest since, according to the Berg mechanism, pyrophosphate does not react until after CoA has dissociated in the back reaction from the E -acetyl-CoA. AMP complex. 6. Steady State Kinetics and Reaction Mechanism
The most detailed kinetic study of the acetate:CoA ligase (AMP) has been made by Farrar and Ploughman (49,60)with partially purified ox lieart enzyme (specific activity 12 pm,oles/mg/min) . Michaelis constants for ATP, acetate, and CoA of 0.71 mM, 0.65 mM, and 0.21 &, respectively, were obtained in 0.1 M tris C1 0.2 M KC1 a t pH 8.3 and 66. W. P. Jencks and J. Carriuolo, JBC 234, 1272 (1957).
482
JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
37", with MgCl, in 0.4 m M excess of ATP; under these conditions the proportion of CoA which is Mg-chelated is nearly constant below 0.5 m M total CoA. Here and below, kinetic results will be discussed in terms of the general initial rate equation for a three substrate reaction enjoying linear kinetics, written according to Dalziel (67):
with A, B, and C standing for the concentrations of ATP, fatty acid, and CoA, respectively. The Berg mechanism is characterized by the and Farrar and Ploughabsence of the last three terms, i.e., +,,,, man reported parallel families of double-reciprocal plots when ATP was varied a t several CoA concentrations and constant acetate, and when acetate was varied a t several CoA concentrations and constant ATP, indicating, respectively, that the terms (+,,c + +,,!JB) and (+,,+abc/A) both approach zero. A triple transfer enzyme substitution mcchanism is excluded by the converging family of plots obtained when ATP was varied a t several acetate concentrations and constant CoA, i.e., +,,I, is positive. The absence of (Pat and +bc excludes quaternary complex mechanisms with either a rapid equilibrium random order or completely compulsory order of addition of substrates ( 6 7 ) .This kinetic pattern is consistent with the Berg mechanism, but also with a partially compulsory order quaternary complex mechanism in which CoA adds first to the enzyme followed by random addition of ATP and acetate ( 6 7 ) . The latter mechanism, which seems quite attractive, requires that be a finite term, but no evidence bearing on the existence of this term is available since all the data were obtained in the presence of a fixed concentration of a t least one suhstrate. Like the enzyme from yeast, enzymes prepared from ox or rabbit heart mitochondria can catalyze formation of acetyl-CoA or ATP from free acetyl adenylate and CoA or PP,, respectively. These reactions are probably fast enough to account for the overall forward and reverse reactions, respectively, although to our knowledge no data concerning the relative rates have been published. In contrast, the synthesis of acetyl hydroxamate from ATP, acetate, and hydroxylamine by both yeast and bcef heart enzymes is accelerated more than 100-fold by the addition of CoA (46, 5 6 ) . Coenzyme A therefore appears to be necessary either for the efficient synthesis or use of the acetate derivative that reacts with hydroxy lamine. Ox heart acetyl-CoA synthetase catalyzes an acetate-potentiated
+
67. K. Dalziel, BJ 114, 547 (1969)
14.
FATTY
ACYL-COASYNTHETASES
483
ATP-3’PPi exchange with a maximum rate, a t pH 8.0, of about 1% of the overall forward reaction rate ( 5 0 ) .The rate of the exchange increases with the acetate concentration up to 1 m M acetate, above which concentration acetate inhibits the reaction in an uncompetitive manner. Farrar interprets thesc data as being consistent with a compulsory order, Berg-type mechanism with A T P as the first substrate. However, some highly purified preparations of ox heart enzyme catalyze an ATP-PPi exchange in the absence of added acetate (61). C. MEDIUMCHAINFATTYACID:COALIGASES(AMP) 1. Substrates and Inhibitors
The broad specitivity of Mahler’s enzyme from ox liver mitochondria, including C,-C,, fatty acids and a variety of aromatic acids, was reviewed by ,Jencks (1). It now appears that both o-methoxylbenzoate and anthranilate can serve as suhstrates for this enzyme although salicylate and p-aminosalicylate cannot ( 1 4 ) .In tris buffer, pH 8, 50 pA4 salicylate, salicylamide, and gentisate inhibit hexanoyl-CoA formation from 0.25 mM hexanoate by 65, 35, and 30%, respectively (68). Mahler’s enzyme can be distinguished from a new “salicy1ate”:CoA ligase (AMP) partially purified from the same source. The latter is active with hexanoate, benzoate, o-methoxybenzoate, and anthranilate as well as with salicylate and p-aminosalicylate (14). Apparent K , values with salicylate as substrate are 1.4 & for salicylate and 14 pLM for ATP (68). This enzyme is strongly stimulated by pyrophosphatase and is stabilized by the combination of salicylate and ATP-Mg ( 1 4 ) . The intermediate chain fatty acid:CoA ligase from ox heart mitochondria shows activity with C,-C, fatty acids including isobutyrate and crotonate but not 0-hydroxybutyrate and acetoacetate ( 1 7 ) .Neither cysteine nor glutathione could substitute for CoA, G T P did not replace ATP, and the slight activities with ITP and C T P may have resulted from contamination with ATP. Unlike Mahler’s enzyme, the ox heart enzyme was not stimulated by pyrophosphatase and was only 50% inhibited by 8 mM pyrophosphate. Apparent K , values were 1.5 mM for butyrate, 3 mM for ATP, and 0.85 mM for CoA. Activation of medium chain enzyme in rat heart by 3‘,5‘-cyclic AMP and some other adenine nucleotides has been suggested (69). 68. P. Killenberg and L. T. Webster, Jr., unpublished observations. 69. V. G. Erwin, A. D. Anderson, and G . J. Eide, J . Pharm. Sn’. So, 77 (1971).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, JR.
2. Formation of Butyryl Adenylate
Butyryl adenylate has been isolated from the trichloroacetic acid supernatant of reaction mixtures containing [“C] butyrate, ATP, MgCl,, and large amounts of Mahler’s enzyme (70). Pyrophosphatase increased the yield of butyryl adenylate from 1.5 pmole/100,000g of enzyme of specific activity 0.28 pmole/min/mg although it did not alter the yield of acetyl adenylate obtained from acetyl-CoA synthetase.
3. Steady State Kinetics and Reaction Mechanism Bar-Tana et al. have carried out a kinetic analysis of the activation of butyrate by their Fractions I and I1 obtained from “Fraction C” of Mahler et a2. (11) by the additional steps of gel filtration and chromatography on DEAE (16). Fraction I1 exhibited linear kinetics which fitted equation (3) for the special case where +ab is absent or very small (71). I n particular, at high concentrations of the third substrate, the slopes of double reciprocal plots with butyrate or A T P as variable substrates increased as the concentration of CoA was decreased, indicating the existence, respectively, of both (+bc + +abC/A) and (+ac + +abc/B). ‘No cnzyme substitution mechanism has an initial rate equation containing either +abc or both +bc and +,,C. The slopes of the double reciprocal plots with butyrate as variable substrate a t constant CoA concentration were the same (parallel lines) over a range of A T P concentrations sufficient to cause a 4-fold increase in the intercept. This shows the probable absence of +&b which excludes a rapid equilibrium random order mechanism and fixes CoA as the middle substrate in a compulsory order quaternary complex mechanism. Apparent Michaelis constants with Fraction I1 a t pH 8.0 (tris) and 37” were ATP, 0.7 m M ; butyrate, 10 mM; and CoA, 0.2 mM. Fraction I proved less amenable to this type of analysis (72). BarTana et al. felt that the results fit an enzyme substitution mechanism of the Berg type. However, because of the small ranges of substrate concentrations and the nonlinearity of some of the double reciprocal plots, the data do not convincingly demonstrate the absence of +bc and +,,be. Since it also seems that Fraction I may be an altered form of Fraction I1 (16),the present reviewers would suggest that a quatcrnary complex mechanism must be seriously considered for both fractions. Apparent Michaelis constants for this fraction were still higher than for Fraction 11: ATP, 6 mM; butyrate, 20 mM; and CoA, 0.4 mM. The high K , 70. L. T. Webster, Jr. and F. Campagnari, JBC 237, 1050 (1962). 71. J. Bar-Tana and G. Rose, BJ 109, 283 (1968). 72. J. Bar-Tans, and G. Rose,BJ 109, 275 (1968).
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values that both fractions exhibited for some of the substrates may indicate some damage to the enzyme. Differences between the partial reactions of Fractions I and I1 of BarTana et d . were also observed. Both fractions catalyze the conversion of butyryl adenylate and CoA to butyryl-CoA a t rates greater than the overall forward reaction. This finding is consistent with the Berg mechanism. With Fraction I but not I1 the dependence of the rate of this partial reaction on the CoA concentration is strongly sigmoidal under certain conditions although results were variable (compare Figs. 8 and 9, ref. 72). In addition, Fraction I catalyzes the synthesis of A T P from butyryl adenylate and PPi, but Fraction I1 only very weakly catalyzes this partial reaction. It is characteristic of all Berg-type mechanisms, in which PPi must dissociate from the enzyme immediately before CoA reacts in the forward reaction, that PPi should be a simple competitive inhibitor of CoA (73). However, Graham and Park ( 7 4 ) , studying the activation of octanoate by Mahler’s enzyme, found that 1 mM PPi caused a 5-fold increase in the intercept of CoA double reciprocal plots without affecting the slope. This uncompetitive inhibition pattern is convincing evidence against a simple Berg mechanism, although the mechanism proposed by the authors must be regarded as highly tentative in view of the fragmentary nature of their data.
D. LONGCHAINFATTY ACID:COALIGASE(AMP) 1. Substrates and Inhibitors Long chain fatty acyl-CoA synthetases of the type first described by Kornberg and Pricer (76) have been difficult to purify and characterize because of their lipid environment and the low aqueous solubility of their substrates. Clearly, the type and concentration of detergent often present in the assay mixtures and the physical state of the fatty acid substrates may be expected to effect greatly the kinetics and substrate specificity of these enzymes. The recent development of an accurate, sensitive assay method based on the insolubility of long chain acyl-CoA’s in diethyl ether allowed Marcel and Suzue to study the kinetics of long chain fatty acid activation by rat liver microsomes a t p H 7.4 a t substrate concentrations below the critical micellar concentrations of both the fatty acids and their acyl-CoA products (27).These investigators found similar low 73. W. W. Cleland, BBA 67, 173 (1963). 74. A. B. Graham and M. V. Park, BJ 111,267 (1969). 75. A. Kornberg and W. E. Pricer, Jr., JBC 204, 329 (1953).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
values for K , (1-2 @ for four I) unsaturated fatty acids having a double bond in the A9 position (palmitoleate, oleate, linoleate, and linolenate) with values of V,,,,, ranging from 9.1 to 18.6 pmoles/hr/mg a t 37". Each of these fatty acids acted as competitive inhibitors of the activation of the others (apparent Ki values of 1.2 &). Palmitate showed a similar pattern ( K , of 2.8 pM; V,,, of 14.3 pmoles/hr/mg; Ki of 1.2 pM and 2.4 pkf against linolenate and palmitoleate, respectively). The apparent K , for CoA was similar for the activation of palmitate (7.2 p M ) and oleate (9.4 pM). The authors concluded that saturated and unsaturated fatty acids were probably activated by the same enzyme. Bar-Tana et al. (B),using older assay methods with material purified from rat liver microsomes (Section I,B,3), found some variations of K,, and V,,, with variations of the chain length and degree of saturation of the fatty acid substrates. At p H 8.0, the specific activity of this preparation with palmitate was 15 pmoles/hr/mg a t 37" and apparent Michaelis constants were ATP, 4.65 m M ; CoA, 50 @; and fatty acids, 11 p.M (C12) and 42 & (Clo), Compared to Marcel and Suzue's intact microsomes, this purified preparation had large K , values for fatty acid substrates and a comparable V,,,,, with palmitate. Because of the differences in methodology, these discrepancies may be apparent rather than real, but they should be recalled when interpreting the investigations of Bar-Tana et al. described below. 2. Partial and Exchange Reactions With the solubilized and purified enzymc (28) described in Section I,B,3, Bar-Tana et al. found that the maximum velocities of both partial reactions (viz, ATP formation and palmitoyl-CoA formation, respectively, from palmitoyl adenylate) were only 1% of that of the overall forward reaction rate ( 7 6 ) . Preincuhation of this enzyme preparation with ATY and CoA did not increase the rate of A T P formation from palmitoyl adenylate, although with an earlier less purified preparation such reactivation of this partial reaction could be shown (77). These partial reactions are readily catalyzed by intact microsomes. It is not yet clear whether their loss during solubilization of palmitoyl-CoA synthetase activity represents a change in the fundamental properties of palmitoyl-CoA synthetase or merely a purification. Both microsomes and the more highly purified enzyme catalyzed the incorporation of 32PYiinto ATP in the absence of all other substrates a t rates of 65% and 6.676, respectively, of thc overall forward reaction. The 76. J. Bar-Tana, G. Rose, and B. Shapiro, BJ 129, 1101 (1972) 77. J. Bar-Tana and €3. Shapiro, BJ 93, 533 (1904).
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significance of this exchange reaction, which is markedly inhibited by 0.2 mM palmitate, is far from clear, especially because small amounts of endogenous fatty acids are likely to be present in preparations derived from microsomes. I n a recent kinetic analysis of the forward reaction of the purified enzyme, Bar-Tana e t al. found that the sums (&,c + +abe/A) and (& + +abc/B) both approached zero, while (+ab + +abc/C) was positive (78). The evidence for, and interpretation of this kinetic pattern, is analogous to that concerning Farrar and Ploughman’s work with acetyl-CoA synthetase (see Section III,B,6), i.e., it is consistent with Berg’s enzyme substitution mechanism but also with a partially compulsory order quaternary complex mechanism. However, interpretation in the present case is also complicated by the fact that palmitate was probably present in the reaction mixtures partly as micelles and mixed micelles containing Triton X-100. Pulse labeling experiments were performed (78) to try to define a “stable” enzyme-substrate intermediate that should occur in any enzyme substitution mechanism (79).However, the results did not fit any simple pattern, and it is doubtful in any case whether pulse labeling experiments distinguish between “stable” and “unstable” enzyme-substrate complexes.
E. CONCLUSIONS Berg’s mechanism was originally proposed for acetyl-CoA synthetase from yeast, but has been widely assumed to describe all fatty acid:CoA ligases (AMP). It contains two distinct and independent postulates: (1) enzyme-bound acetyl adenylate is an obligatory intermediate and (2) PPi must dissociate from the enzyme immediately before CoA reacts in the forward reaction. There is good evidence that several ligases can make and use acyl adenylates. Enzyme-bound acetyl adenylate and butyryl adenylate can be isolated from reaction mixtures containing the respective activating enzymes. Yeast and heart acetyl-CoA synthetases, and possibly the microsomal long chain enzyme, catalyze fatty acid-dependent, CoAindependent [“PPi 1ATP exchanges. The acetyl-CoA synthetases, the medium chain enzyme of liver mitochondria, and some, but not all, preparations of microsomal long chain enzyme utilize acyl adenylates in partial reactions. However, these observations do not show that the acyl adenylates are obligatory intermediates. For all three enzymes, these 78. J. Bar-Tana, G. Rose, R. Brandes, and B. Shapiro, BJ 131, 199 (1973). 79. W.W.Cleland, BBA 67, 104 (1963).
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JOHN C. LONDESBOROUGH AND LESLIE T. WEBSTER, J R .
and other properties depend on the method of preparation. Thus, the recent high specific activity acetyl-CoA synthetase (Section I,B,l) can make only very small amounts of enzyme-bound acetyl adenylate, the medium chain enzyme can be resolved into varying proportions of two fractions, only one of which efficiently catalyzes the conversion of butyryl adenylate to ATP, and the long chain enzyme loses all partial reactions involving acyl adenylate during its purification. It is even possible that these partial reactions and the formation of enzyme-bound acyl adenylates are not inherent properties of the native enzymes. The second postulate of Berg’s mechanism, namely, that PPi must dissociate immediately before CoA binds, leads to kinetic predictions which are, in principle, easy to test. The reciprocal initial rate equation should contain no terms with both CoA and another substrate in the denominator, i.e., +8c = +bc = +abc = 0. The product inhibition pattern is also useful to identify this mechanism. Unfortunately, partly because of the instability of the various enzymes but mainly because there is no simple accurate assay procedure (see Section III,A), kinetic data of sufficient quality to show the absence of initial rate parameters are difficult to obtain. So far no study has been made in which low concentrations of all three substrates have been used in an attempt to test the existence of +abc. For ox heart acetyl-CoA synthetase, and possibly microsomal palmitoyl-CoA synthetase from rat liver, the kinetic data are consistent with Berg’s mechanism, but also with a partially ordered quaternary complex mechanism with CoA as the leading substrate and ATP and fatty acid subsequently adding randomly. For the medium chain enzyme, inhibition by PPi is not of the simple competitive type as required by the Berg mechanism. Furthermore, intersecting patterns of double reciprocal plots result when the concentrations of either ATP or butyrate are varied a t several CoA concentrations. These observations show that for certain preparations of medium chain acyl-CoA synthetase, PPi does not dissociate immediately before addition of CoA (Section IXI,C,3). The long chain enzyme solubilized from rat liver seems to require bound CoA for the expression of any activity (Section III,D,2). It therefore appears that dissociation of PPi may not precede reaction with CoA and therefore a quaternary eneyme-substrate complex occurs. This opens up the possibility for concerted mechanisms, although the transitory occurrence of acyl adenylates in the central enzyme-substrate complexes cannot be excluded by initial rate data. A satisfactory mechanism has not been convincingly established for any fatty acid:CoA ligase (AMP), and the information presently available suggests there may be important mechanistic differences between the members of this class of enzymes.