Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3458-3462, September 1975 Biochemistry Isolation of subunits of citrate and characterization of their function in the complex (citryl-S-acyl carrier protein/) P. DIMROTH AND H. EGGERER Fachbereich Biologie der Universitat, Regensburg, BRD-8400 Regensburg, Universititsstrafle 31 Communicated by F. Lynen, July 7, 1975

ABSTRACT Citrate lyase [EC 4.1.3.6; citrate oxaloace- unit, the a-chain, in the presence of acetyl-S-acyl carrier tate-lyase (pro-3S-CH2COO- -_ acetate)] from Klebsiella aer- protein and citrate, catalyzes the formation of citryl-S-acyl ogenes has been dissociated with urea; the three different carrier protein plus acetate and that the other subunit, the subunits, a-chain (molecular weight 54,000), f-chain (mo- lecular weight 32,000), and 'y-chain (acyl carrier protein; fl-chain, catalyzes the cleavage of this citryl derivative to the molecular weight - 10,000), have been isolated in pure and acetylated species with liberation of oxaloacetate. It is tenta- catalytically active state. Recombination of the three sub- tively suggested, therefore, to name these citrate units produced citrate lyase that was indistinguishable from acyl carrier protein transferase (a-chain) and citryl-S-acyl the untreated enzyme. The a-chain in the presence of acetyl- carrier protein lyase (f-chain). S-acyl carrier protein catalyzed the formation of the corre- sponding citry thioester with liberation of acetate, and the a-chain catalyzed the cleavage of citryl-Sacyl carrier protein MATERIALS AND METHODS with liberation of oxaloacetate. A simple enzymic method Citrate lyase of initial specific activity about 50 U/mg of for the preparation of citryl-S-acyl carrier protein is de- protein was prepared from K. aerogenes as described (3). scribed. On storage the enzyme lost enzymic activity due to deacety- lation; partially deacetylated specimens were used for the Citrate lyase [EC 4.1.3.6; citrate oxaloacetate-lyase (pro-3S- separation procedure described below. CH2TCOO - acetate)] from Klebsiella aerogenes catalyzes Resolution of Citrate Lyase Subunits. Citrate lyase (120 the cleavage of citrate to acetate and oxaloacetate (Eq. 1) mg) was incubated for 2 hr at 00 in a total volume of 6 ml of and represents an 10 mM phosphate buffer (K+), pH 7.0, containing 5 M urea. The solution was applied to a column of DEAE-cellulose (2 citrate t acetate + oxaloacetate [11 X 6 cm) that had been equilibrated with 10 mM phosphate buffer, pH 7.0, containing 4 M urea. Elution of the protein- enzyme complex that consists of three different polypeptide loaded column by use of this buffer/urea solution yielded a chains, a, (3, and y, with molecular weights about 54,000, protein-containing eluate (fraction 1), which appeared with- 32,000, and 10,000, respectively (1-3). Six copies of each of in the first 25 ml. After a total of 50 ml of eluate was collect- these subunits appear to be arranged in the complex with ed, urea was removed from the column by washing with 50 formation of a hexameric structure, (a, (, ')6 (3). The y- ml of 10 mM phosphate buffer (K+), pH 7.0. Subsequent chain was shown to be an acyl carrier protein that carries an elution performed with 50 ml of the same buffer but in the acetyl group in thioester linkage to a cysteamine residue (1). presence of 0.4 M NaCl produced another protein-contain- This is part of the prosthetic group consisting of a substituted ing eluate (fraction 2). This was followed by elution with 50 dephospho-CoA isomer (4). ml of the same buffer but in presence of 0.4 M NaCl and 4 The overall mechanism of action of the enzyme involves M urea to yield fraction 3. All urea-containing buffer solu- the consecutive reactions of acyl exchange (Eq. 2) and acyl tions were freshly prepared prior to use. cleavage (Eq. 3) (5, 6). Immediately after the separation the three fractions were treated at 40 as follows. Fraction 1, containing the a-chain acetyl-S-enzyme + citrate was dialyzed for 16 hr against 1 liter of 0.5 M Tris-HCl buff- er, pH 8.0, 10 mM in mercaptoethanol, and then for another citryl-S-enzyme + acetate [2] 24 hr against 1 liter of 0.1 M Tris-HCl buffer, pH 8.0, 1 mM in dithioerythritol. Dialysis was preceded by dilution of citryl-S-enzyme fraction 1 with 10 mM phosphate buffer (K+), pH 7.0, 4 M acetyl-S-enzyme + oxaloacetate. [3] in urea, to a protein concentration of 1 mg/ml. The a-chain was homogeneous [ultracentrifuge (3)]. Since two chemically different reactions participate in the The Ey-chain present in fraction 2 was dialyzed and puri- cleavage of citrate and since the enzyme contains two differ- fied to homogeneity (manuscript in preparation; see ref. 1); ent proteins in addition to the acyl carrier protein, it was it was only partially acetylated. Partial deacetylation took tempting to speculate that one of these would be responsible place on storage of citrate lyase and also, as described (1), for the acyl exchange reaction (Eq. 2), the other for the acyl during isolation of the y-chain. Fraction 3, containing the lyase reaction (Eq. 3) (1). Conclusive evidence was missing, fl-chain, was dialyzed for 16 hr against 1 liter of 10 mM however, because the demonstration of different enzymes as phosphate buffer (K+), pH 7.0, 1 mM in dithioerythritol and constituents of the complex had to await their isolation in 1 mM in MgCl2. Dialysis was continued for another 24 hr pure and catalytically active states. This gap is filled by the against 1 liter of fresh dialysis medium. Fraction 3 was results presented in this paper. It will be shown that one sub- found to be free of the a-chain but contained small amounts 3458 Downloaded by guest on September 27, 2021 Biochemistry: Dimroth and Eggerer Proc. Nat. Acad. Sci. USA 72 (1975) 3459

..,:wdtfr

, _

FRACTION FIG. 1. Resolution of citrate lyase subunits on Dl:AR-cellu- lose. For details see text. Absorbances were determined at 260 nm (0) and 280 nm (0). - A B C D

sk (about 15% in molar relationship) of acyl carrier protein. i This impurity was removable by chromatography on Sepha- ..-w dex G-75 (1 X 78 cm; 0.05 M phosphate buffer, pH 7.0, 10 FIG. 2. Dodecyl sulfate/polyacrylamide gel electrophoresis of mM mercaptoethanol). It was found consistently, however, citrate lyase and the isolated subunits. Preparation of samples and that this pure f-chain rapidly lost enzymic activity, as of 10% polyacrylamide gel and electrophoresis were performed as judged from the results of reconstitution described (7). (A) Separated protein bands of citrate lyase; (B) iso- experiments. lated a-chain (fraction 1); (C) isolated y-chain (fraction 2 after Therefore, in most of the studies presented in this paper, not further purification); and (D) isolated ,8-chain (fraction 3 after the pure A-chain but samples contaminated with the y-chain chromatography on Sephadex G-75). were used. Recombination of Citrate Lyase from the Isolated Sub- units. The three subunits in a total volume of 0.1 ml were scribed under Materials and Methods. The results are preincubated for 20 min at 250 in Tris-HCl buffer, pH 8.0, shown in Fig. 1. The recovery of protein present in the three containing 5 mM MgCl2 and 10 mM dithioerythritol. A solu- fractions (33, 16, and 17 mg, respectively) was 55% of the tion (0.85 ml) containing 100 gimol of Tris-HCl buffer, pH total applied to the column. The separation into three pro- 8.0, 1 Amol of MgCl2, and 0.3 limol of NADH was then tein fractions suggested a resolution of the enzyme complex added. The reaction mixture was treated with 10 jA of 0.1 M into the three subunits that have been observed on dodecyl acetic anhydride (in dioxane) for conversion of deacetyl ci- sulfate/disc gel electrophoresis (1-3). This method was used trate Iyase into the acetylated species. Malate dehydrogenase for analysis of the three protein fractions by comparing their (EC 1.1.1.37) (20 U) and citrate (20 Ai; 2 Mmol) were then mobilities with those of the three protein bands obtained added and citrate Iyase activity was determined without from a parallel run of the unfractionated enzyme complex. delay (5). The results (Fig. 2) demonstrate that the three subunits of ci- Enzymic Formation of [1,5-14C2JCitryl-S-Acyl Carrier trate lyase were in fact separated by the DEAE-cellulose Protein. The incubation mixture, in a total volume of 1.0 ml, chromatography. The protein that was not adsorbed to the contained 50 mM Tris buffer (pH 8.0), 0.55 mM [1,5- column (fraction 1) consisted of the a-chain (molecular 14C2]citrate (3 X 107 cpm X MmoI-'), 1.3 mg of acetyl-S- weight 54,000); fraction 2 contained the y-chain (molec- acyl carrier protein (a partially acetylated specimen), and 60 ular weight - 10,000); and fraction 3 the f-chain (molecu- ,gg of a-chain. The reaction was initiated by adding the lat- lar weight E 32,000). ter enzyme, and after a 40-min incubation at 250 the reac- The a-chain, that was enzymically active (see below), had tion mixture was applied to a column of Sephadex G-75 Su- a molecular weight of about 110,000, as judged from high- perfine (1 X 78 cm) which was kept at 40. Elution was per- speed sedimentation equilibrium centrifugation performed formed with 5 mM phosphate buffer (K+), pH 7.0. in Tris buffer, pH 8, as well as from gel filtration experi- Protein was determined by the biuret method, with the ments. It is, therefore, a dimer of a-chains. Whether these exception of the acyl carrier protein which was determined a2-units remain structurally intact on incorporation into the from the absorbance at 260 nm (4). Radioactivity was deter- complex remains to be established. However, recent results, mined by liquid scintillation counting. which were obtained by electron microscopic studies with citrate lyase from Rhodopseudomonas gelatinosa, are con- RESULTS sistent with dimers of subunits as structural element of this Isolation of citrate lyase subunits lyase (9). Previous work established the dissociation of citrate Iyase by Reconstitution of citrate lyase from isolated subunits 5 M urea into subunits from which one, the acyl carrier pro- None of the isolated subunits was enzymically active in the tein, could be isolated in a homogeneous state (1). Attempts cleavage of citrate to acetate and oxaloacetate (Eq. 1), as ex- to improve this method with the aim of obtaining the y- pected, and no enzymic activity in the cleavage of citrate chain in larger amounts (P. Dimroth, manuscript in prepara- could be observed by simply mixing the urea-containing tion) led to the isolation of the three lyase subunits as de- subunit solutions. Dilution experiments (up to about 10-fold) Downloaded by guest on September 27, 2021 3460 Biochemistry: Dimroth and Eggerer Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Recombination of citrate lyase from isolated subunits Citrate lyase activity Omissions (mU) None 636 Xx-Chain 0 13-Chain 0 7y-Chain 90 The experiments were performed as described in Materials and Methods with 23 gg of a-chain, 16 ,g of d3-chain (fraction 3), and 1.3 gg of ey-chain.

strable. The results indicate that the a- and y-chains could TIME (min) be completely renatured by dialysis but that about 80% of FIG. 3. Kinetics of citrate lyase formation from the isolated enzymic activity of the 13-chain was probably irreversibly subunits. The incubation mixture, which was kept at 250 in a total lost during the isolation and renaturation. volume of 1.5 ml, contained 0.1 M Tris.HCl buffer (pH 8.0), 5 mM The reconstituted enzyme was identical with the native MgCl2, 10 mM dithioerythritbl, 220 uig of a-chain, 380 ,g of fl- enzyme complex as judged from identical sedimentation chain, and 30 ;ig of Ey-chain. The reaction was initiated by adding coefficients determined by centrifugation in sucrose density the a-chain. Samples (0.1 ml) were withdrawn at the times indicat- ed and the activity of citrate lyase was determined as described in gradients. The isolated, reconstituted complex was well sep- Materials and Methods. arated from excess protein components; its specific activity was as high as that of native citrate Iyase; i.e., 55 U/mg of protein. were also inefficient. However, dialysis of the subunit frac- tions under proper conditions (see Materials and Methods) Characterization of a-chain as citrate acyl carrier caused renaturation and produced catalytically active citrate protein transferase lyase in recombination experiments. The conditions for re- When [1,5-14C2]citrate was incubated with unlabeled acetyl- naturation by dialysis were found to be especially critical for S-acyl carrier protein either alone or in presence of the iso- the a-chain, where a pH of 8.0, high ionic strength (0.5 M lated 13-chain, the acyl carrier protein remained unlabeled. Tris buffer), and the presence of mercaptans as well as a If, however, the a-chain was substituted in parallel experi- protein concentration of about 1 mg/ml were found to be ments for the 1-chain, [1,5-'4C2]citryl-S-acyl carrier protein optimal. was formed by acyl exchange (Fig. 4). This result not only If the dialyzed protein fractions 1-3 were combined, the demonstrates that the a-chain is responsible for catalysis of activity of citrate lyase was generated in a time-dependent the acyl exchange reaction, but also provides a simple means process. This was influenced by subunit concentration, the to prepare citryl-S-acyl carrier protein. The formation of absence or presence of dithioerythritol, buffer concentra- this compound increased initially and reached a plateau tion, and temperature. The result of a representative recom- after about 20 min of incubation, when 3.9 nmol (25% yield) bination experiment under optimal conditions is shown in were formed (Fig. 4). This low yield reflects a partial acety- Fig. 3. lation of the acyl carrier protein (see Materials and Meth- Citrate lyase formation in these reconstitution experi- ods). ments required the presence of all three subunits (Table 1), The specific activity for acyl exchange in the experiment as expected. Incubation of any fraction alone or of only the of Fig. 4 is about a hundred times lower than calculated for Ey-chain (fraction 2) with the a-chain (fraction 1) or the 13- the same amount of enzyme integrated in the enzyme com- chain (fraction 3) failed to produce catalytic activity for the plex. This result indicates that formation of the complex cleavage of citrate. A small amount of citrate lyase activity greatly enhances the catalytic power of the individual sub- was produced, however; on combination of fractions 1 and 3 units. in the absence of fraction 2. This was consistently observed Characterization of 1-chain as citryl-S-acyl carrier and could be traced by disc gel electrophoretic analysis to protein Iyase the presence of small amounts of acetyl-S-acyl carrier pro- tein impurity in fraction 3. The impurity was removable In these experiments, the lyase-dependent cleavage of [1,5- (see Fig. 2), but the lyase was then unstable (see Materials '4C2]citryl-S-acyl carrier protein was followed from the dis- and Methods). From the previously given correlation of tribution of radioactivity (see Materials and Methods). subunit composition (1:1:1) and enzymic activity of the en- When [1,5-14C2Jcitryl-S-acyl carrier protein was incubated zyme complex (3) the percentage of reconstitution was cal- with the isolated 13-chain (fraction 3), cleavage of the citryl culated to be about 50 and 64%, respectively, from the data thioester to the acetylated derivative took place with libera- of Table 1 and Fig. 3, if corrected for the presence of acyl tion of [4-'4C]oxaloacetate, which was reduced enzymically carrier protein as an impurity in fraction 3 containing the to malate (Table 2, Exps. 1 and 2). About half of the total ra- 13-chain. A higher degree of reconstitution, up to 100%, was dioactivity appeared in malate and the other half was bound achieved if either the a-chain or the y-chain was used in to protein, as one would expect upon complete cleavage of limiting amounts in the presence of an excess of each of the the to yield [1-'4C]acetyl-S-acyl carrier protein two other subunits. If, however, the 1-chain was used in lim- and oxaloacetate. No cleavage reaction was detectable if the iting amounts in such experiments, then only about 20% of 1-chain was omitted (Exp. 3) or it it was replaced by the ay- the theoretically expected citrate lyase activity was demon- chain. However, citrate was slowly liberated in the presence Downloaded by guest on September 27, 2021 Biochemistry: Dimroth and Eggerer Proc. Nat. Acad. Sci. USA 72 (1975) 3461

of this subunit (Exp. 4), whereas no reaction would be ex- pected under these conditions. The a-chain may thus exhibit a hydrolytic activity towards citryl-S-acyl carrier protein, 0 and this could account for the reaction inactivation of citrate LII 0o S lyase (10, 11). It is more likely, however, that this transfer- ~ ase-dependent apparent hydrolysis stems from trace z

amounts of acetate present in the incubation mixture. The 0 citryl-S-acyl carrier protein (about 5 nmol) would react with LL the impurity in the presence of the a-chain to yield the un- 1.0- labeled acetylated derivative and [1,5-14C2]citrate. This re-

versal of formation of citryl-S-acyl carrier protein took place a.- rapidly, as expected, when acetate was added (Exp. 5). The .0 partial loss of radioactivity (see "Total" in Table 2) is proba- bly due to loss of labeled acyl carrier protein during the ana- L> O-0 lytic procedure. In summary, the results show the biological activity of citryl-S-acyl carrier protein in the cleavage reac- 0 10 20 30 tion and characterize the fl-chain as the enzyme complex TIME (min) subunit responsible for the partial reaction of acyl cleavage FIG. 4. Kinetics of enzymic formation of [1,5-14C2]citryl-S- (Eq. 3). acyl carrier protein. The incubation mixture, which was kept at 25° in a total volume of 1.0 ml, contained 0.1 M Tris-HCl buffer DISCUSSION (pH 8.0), 0.11 mM [1,5- 4C2]citrate (3 X 107 cpm X gmol-1), 0.16 mg of acetyl-S-acyl carrier protein (partially acetylated), and 12 tg It has been shown previously that deacetyl citrate lyase and of a-chain, which was used to initiate the reaction. Samples (50 Ml) its carboxymethylated derivative had lost the ability to cata- were withdrawn at the times indicated, and the formation of [1,5- lyze the cleavage of citrate (Eq. 1). In the presence of acetyl- 14C2]citryl-S-acyl carrier protein was determined from radioactivi- CoA, however, these inactivated enzyme specimens cata- ty in trichloroacetic acid-precipitated protein. lyzed the cleavage of citrate according to Eqs. 4 and 5 via

intermediate formation of 3S-citryl-CoA (6). - citryl-S-acyl carrier protein --

acetyl-CoA + citrate W citryl-CoA + acetate [4] acetyl-S-acyl carrier protein + oxaloacetate [r7 However, conclusive evidence was missing because only the citryl-CoA acetyl-CoA + oxaloacetate [51 acetyl-S-acyl carrier protein and not the corresponding ci- in enzyme. Since acetyl-CoA had a catalytic function in this system, it tryl thioester could be isolated work with the it was enzymic activities was concluded that both acetyl-CoA and citryl-CoA could Moreover, unknown whether the substitute for the native enzyme-bound intermediates by involved in cleavage of citrate on the enzyme would be in- virtue of structural similarities (6). This suggestion was sub- herent properties of the individual subunits or would arise stantiated by isolation of the acyl carrier protein (1) and elu- only upon their assembly into the organized complex. cidation of the structural similarities of its prosthetic group These ambiguities are resolved by the results presented in with coenzyme A (4). It naturally follows that the acetyl-S- this paper. It is clear now that the enzyme complex is com- acyl carrier protein of the enzyme complex reacts with ci- posed of the acyl carrier protein and two different enzymes, trate to yield 3S-citryl-S-acyl carrier protein (Eq. 6) and that a transferase and a lyase, which catalyze the reactions of this intermediate is subsequently cleaved to the acetylated Eqs. 6 and 7 during citrate degradation in microorganisms. derivative with liberation of oxaloacetate (Eq. 7). It is also apparent that the acylated prosthetic group of the acyl carrier protein must oscillate during catalytic action be- acetyl-S-acyl carrier protein + citrate tween the different enzyme subunits within the complex. Several advantages have been considered for multien- citryl-S-acyl carrier protein + acetate [6] zyme cQmplexes compared to solutions of their separate en-

Table 2. Enzymic cleavage of [1,5-14C2]citryl-S-acyl carrier protein Radioactivity (cpm) Bound Exp. Addition to protein In acetate In malate In citrate Total 1 None 42,000 0 0 0 42,000 2 3-Chain (140 pg) 22,800 0 18,100 0 40,900 3 j3-Chain (280 Mg) 19,700 0 20,100 0 39,700 4 a-Chain (60 pg) 8,600 0 0 46,000 54,600 5 ca-Chain (24 Mg) + 1,600 0 0 45,000 46,600 acetate (1 pmol) The incubation mixtures, which were kept at 250 in a total volume of 1.0 ml, contained 50 mM Tris.HCl buffer, pH 8.0; 2 mM MgC12; 0.3 mM NADH; 100 U of malate dehydrogenase; 5.1 MM [1,5-14C2]citryl acyl carrier protein (52 000 cpm), and the subunits of citrate lyase as listed in the table. The reactions were started by addition of [1,5-14C2]citryl-S-acyl carrier protein, and terminated after 15 min by adding 0.1 ml of 3 M HCl; 0.5 Mmol of malate and 0.5 Mmol of citrate were added as carrier and the precipitated protein was centrifuged off. The radio- activity of the reaction products was determined after separation by Dowex-1 chromatography (8). Downloaded by guest on September 27, 2021 3462 Biochemistry: Dimroth and Eggerer Proc. Nat. Acad. Sci. USA 72 (1975) zymes (12). The observation that the rate of formation of ci- resembles citrate lyase and which represents a group I en- tryl-S-acyl carrier protein in the presence of the isolated a- zyme. chain is about a hundred times slower than the overall reac- In summary, the structural and functional aspects of ci- tion (Eq. 1) is consistent with these proposals. trate lyase fit well into those of other enzyme complexes. It is interesting in this context to note that the a-chain is a The special feature of the citrate and citramalate ap- catalytically-active dimer, not a monomer. Whether the mo- pears to be a "built-in catalyst," the acetyl thioester group. nomeric a-chain is enzymically active at all is yet unknown. Once this is formed enzymically (23) from the deacetyl en- The same relationship may apply for the (-chain, for which zyme, it allows continuous substrate turnover with regenera- corresponding determinations have not been made. The ob- tion of new acetyl-S-enzyme (Eqs. 2 and 3). Such a reaction servation that the (3-chain rapidly becomes inactivated if iso- sequence, to our knowledge, is unique in enzymology. lated in pure state, free of the y-chain, may indicate its sta- bilization by We thank Prof. J. R. Mohrig for reading the manuscript, and protein-protein interactions with the y-chain. Miss R. Loyal and Mrs. B. Melzi for competent technical assistance Similar to the reaction with deacetyl citrate Iyase, the isolat- in part of this work. The molecular weight of the a-chain dimer ed a-chain catalyzed the acyl exchange between acetyl-CoA was kindly determined by Dr. H. Durchschlag by high-speed sedi- and citrate (Eq. 4). The isolated $-chain, however, was inac- mentation equilibrium studies. Support of this work by the Fonds tive in the cleavage of citryl-CoA (Eq. 5). The cleavage of der Chemischen Industrie is gratefully acknowledged. citryl-CoA on the d-chain only took place if the isolated a- 1. Dimroth, P., Dittmar, W., Walther, G. & Eggerer, H. (1973) chain was added to the incubation mixture. This could arise Eur. J. Biochem. 37,305-315. from interactions between the a- and f-chains or from the 2. Singh, M., Carpenter, D. E. & Srere, P. A. (1974) Biochem. formation of deacetyl citrate lyase due to the y-chain impu- Biophys. Res. Commun. 59, 1211-1218. rity present in the f-chain solution. In any case, the observa- 3. Dimroth, P. & Eggerer, H. (1975) Eur. J. Biochem. 53, 227- tions indicate drastic modifications of catalytic power upon 235. assembly of the organized complex from its subunits. 4. Dimroth, P. (1975) FEBS Lett. 51, 100-104. Since citrate lyase contains it resembles 5. Buckel, W., Buschmeier, V. & Eggerer, H. (1971) Hoppe Seyl- pantothenate, er's Z. Physiol. Chem. 352, 1195-1205. gramicidin S synthetase (13), 6-methylsalicylic acid synthe- 6. Buckel, W., Ziegert, K. & Eggerer, H. (1973) Eur. J. Biochem. tase (14), and the fatty acid synthetases (15, 16). However, 37,295-304. the protein composition of the latter complexes, which cata- 7. Weber, K., Pringle, J. R. & Osborn, M. (1972) in Methods in lyze multistep sequences, is different from that of the lyase. Enzymology, eds. Hirs, C. H. W. & Timasheff, S. N. (Aca- Moreover, fatty acid synthetase from yeast consists of only demic Press, New York), Vol. 26, pp. 3-27. two polypeptide chains, each of which contains several en- 8. Cornforth, J. W., Redmond, W., Eggerer, H., Buckel, W. & zymic activities, and one of which contains the pantothenate Gutschow, C. (1970) Eur. J. Biochem. 14, 1-13. residue (17). The fatty acid synthetase from Escherichia coli 9. Beuscher, N., Mayer, F. & Gottschalk, G. (1974) Arch. Micro- contains a carrier protein and individual enzymes but has biol. 100,307-328. as 10. Dagley, S. & Dawes, E. A. (1955) Biochim. Biophys. Acta 17, never been obtained a complex (16). Citrate Iyase can be 177-184. regarded as the simplest of the pantothenate-containing en- 11. Singh, M. & Srere, P. A. (1971) J. Biol. Chem. 246, 3847- zyme complexes but is in fact more related to the bacterial 3850. biotin enzymes acetyl-CoA carboxylase (EC 6.4.1.2) (18) and 12. Ginsburg, A. & Stadtman, E. R. (1970) Annu. Rev. Biochem. methylmalonyl-CoA pyruvate transcarboxylase (EC 2.1.3.1) 39,429-472. (19). These, like the lyase, catalyze a two-step sequence and 13. Lipmann, F. (1973) Acc. Chem. Res. 6,361-367. are composed from a carrier protein and two different en- 14. Dimroth, P., Greull, G., Seyffert, R. & Lynen, F. (1972) zymes. The resemblance is particularly apparent with the Hoppe Seyler's Z. Physiol. Chem. 353, 126. latter enzyme which, like the lyase, has recently been recon- 15. Lynen, F., Oesterhelt, E., Schweizer, E. & Willecke, K. (1968) stituted from the active subunits in Cellular Compartmentalization and Control of Fatty Acid catalytically (19). Metabolism (Universitetsforlaget, Oslo), pp. 1-24. In both the fatty acid synthetases and the biotin enzymes 16. Prescott, D. J. & Vagelos, P. R. (1972) Adv. Enzymol. 36, several groups of functional enzymes can be distinguished 269-311. according to their structures. Thus, the biotin enzymes com- 17. Schweizer, E., Kiep, B., Castorph, H. & Holzner, U. (1973) prise three groups ranging from three different polypeptide Eur. J. Biochem. 39,353-362. chains in group I to only one chain into which the three sub- 18. Lane, M. D., Moss, J. & Polakis, S. E. (1974) in Current Topics units are incorporated (group III) (20). It is interesting to in Cellular Regulation, eds. Horecker, B. L. & Stadtman, E. note that a similar relationship may hold for the citrate lyas- R. (Academic Press, New York), Vol. 8, pp. 139-187. es from different microorganisms. In contrast to the lyase 19. Wood, H. G., Ahmad, F., Jakobson, B., Chuang, M. & Brattin, from K. aerogenes, a group I citrate lyase if the biotin en- W. (1975) J. Biol. Chem. 250, 918-926. is 20. Lynen, F. (1974) in Lipmann Symposium, ed. Richter, D. zyme classification (20) applied, that from R. gelatinosa (Walter de Gruyter, Berlin & New York), pp. 671-698. consists of only two polypeptide chains, one of which con- 21. Buckel, W. (1975) Hoppe Seyler's Z. Physiol. Chem. 356, tains the pantothenate residue (9). This enzyme, therefore, 223-224. represents a group II citrate Iyase. Another related enzyme 22. Martinoni, B. & Arigoni, D. (1975) Chimia 29,26-27. complex is citramalate lyase (EC 4.1.3.22), which in protein 23. Schmellenkamp, H. & Eggerer, H. (1974) Proc. Nat. Acad. composition (21) and mechanism of action (21, 22) strongly Sci. USA 71, 1987-1991. Downloaded by guest on September 27, 2021