Acetyl CoA Carboxylase: The Purified Transcarboxylase Component

Proc. Nat. Acad. Sci. USA Vol. 68, No. 6, pp. 12591263, June 1971 Acetyl CoA Carboxylase: The Purified Transcarboxylase Component (acyl CoA binding/carboxylation/exchange reactions/biotin) ALFRED W. ALBERTS, STUART G. GORDON, AND P. ROY VAGELOS Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Mo. 63110 Communicated by Konrad Bloch, March 31, 1971 ABSTRACT Acetyl CoA carboxylase of Escherichia coli has been resolved into three functionally dissimilar proteins: (1) biotincarboxyl carrier protein (BCCP); (2) a biotin carboxylase component that catalyzes the Mn ATPdependent carboxylation of BCCP to form CO2 BCCP; and (3) a transcarboxylase component that catalyzes the transfer of the carboxyl group from CO2BCCP to acetyl CoA to form malonyl CoA. The transcarboxylase has been purified 1700fold. Evidence that this protein catalyzes the transcarboxylase step includes the demonstration that it (a) catalyzes the carboxylation of BCCP, (b) catalyzes the BCCPdependent exchange between 1l4C]acetyl CoA and malonyl CoA, (c) binds labeled acetyl CoA and malonyl CoA, and (d) catalyzes the decarboxylation of CO2 BCCP. On the basis of this evidence, it is concluded that the transcarboxylase component contains sites for the acyl CoA group and for biotin, the covalently bound prosthetic group of BCCP. Acetyl CoA carboxylase from Escherichia coli is composed of three functionally dissimilar proteins, biotin carboxylase, biotincarboxyl carrier protein (BCCP), and a transcarboxylase component, formerly referred to as Eb (1, 2). These proteins have been shown to function in reactions 13. Biotin carboxylase, Mn2+ ATP + HC03 + BCCP _ C02BCCP + ADP + Pi (1) Transcarboxylase C02 BCCP + CHXCOSCoA t 02CCH2COSCoA + BCCP (2) Sum: ATP + HC03 + CH3COSCoA = 02CCH2COSCoA + ADP + Pi (3) Biotin of BCCP is carboxylated in reaction 1 to form the carboxybiotin derivative, and this reaction is catalyzed by another protein, biotin carboxylase. The roles of BCCP and biotin carboxylase in this reaction have been delineated by Alberts, Nervi, and Vagelos (2). BCCP, which contains covalently bound biotin, has been purified in two forms (3, 4). A large form, BCCPL, has one mole of biotin per 20,000 grams of protein; it is apparently composed of one peptide (4). BCCPL has a Km of about 24 X 10O7 M in reactions 1 and 2. A small form, BCCPs, has one mole of biotin per 10,000 grams of protein and a Km 50100 times higher than BCCPL in reactions 1 and 2 (3, 4). Although the relationship between the different forms of BCCP has not been completely elucidated, we suspect that BCCPs is formed as a result of proteolytic Abbreviation: BCCP, biotincarboxyl carrier protein. 1259 cleavage of BCCPL. Both BCCPs (3) and biotin carboxylase (5) have been crystallized. A third protein, the transcarboxylase component, catalyzes the transfer of the carboxyl group from carboxybccp to acetyl CoA to form malonyl CoA in reaction 2 (1). Its function was initially demonstrated in enzyme preparations that contained both BCCP and biotin carboxylase; it was shown that the transcarboxylase was required in reaction 2 but was not required in reaction 1. The present communication describes the purification of the transcarboxylase component of E. coli acetyl CoA carboxylase. The availability of this protein and BCCP has permitted the unequivocal demonstration that, in the absence of biotin carboxylase, this protein catalyzes reaction 2, the transcarboxylase step of the acetyl CoA carboxylase reaction. In the absence of acetyl CoA, the transcarboxylase catalyzes the decarboxylation of C02BCCP. transcarboxylase C02BCCP + H20 ' HCO3 ± BCCP (4) EXPERIMENTAL PROCEDURE E. coli B cells, grown in rich medium through 75% of the logarithmic phase, were obtained from Grain Processing Corp., Muscatine, Iowa. Acetyl CoA and [214C]acetyl CoA were synthesized (6) as was malonyl CoA (7). [1,314C]Malonyl CoA, [214C]malonyl CoA, and [2'4C]acetate were obtained from New England Nuclear Corp. BCCPS was a generous gift of Dr. A. M. Nervi. Acetyl CoA carboxylase activity and protein were measured as was described (3). Analytical disc gel electrophoresis was performed by the method of Davis (8). Gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate followed the method of Weber and Osborn (9). Purification of Ea All the following steps were performed at 4 C. 1 kg (wet weight) of E. coli B in 0.02 M potassium phosphate (ph 7.0) 1 mm EDTA0.05 M 2mercaptoethanol was homogenized in a MantonGaulin Submicron Disperser, and the cellular debris was removed by centrifugation. Solid ammonium sulfate was added to 45% saturation. The resulting precipitate was dissolved in 0.05 M imidazole HCl (ph 6.7) to a protein concentration of 20 mg/ml. The remaining insoluble material was removed by centrifugation and the supernatant was brought to 45% saturation with ammonium sulfate. The precipitate that formed was recovered by centrifugation and dissolved in a minimal volume of 0.05 M imidazole HCl (ph 6.7). E. activity was adsorbed onto alumina Cy gel at a geltoprotein ratio of 1: 25. After it was stirred for 5 min, the gel was

1260 Biochemistry: Alberts et at. Proc. Nat. Acad. Sci. USA 68 (1971) TABLE 1. Purification of Ea* and Ebt Fold Unitst/mg purified Ea Eb Ea Eb Cell extract 0.0007 0.0033 045% Ammonium sulfate I 0.0017 0.0084 2.4 3.7 045%Ammoniumsulfatell 0.0029 0.0138 4.1 6.0 Alumina Cy supernatant 0.0002 0.142 6.2 Alumina CGy eluate 0.291 0 410 Hydroxylapatite eluate 0.164 72.0 Sepharose4B 0.60 260 Ammonium sulfate extraction 1.63 710 DEAEcellulose 3.95 1700 10 f Fig. 1. Carboxylation of BCCPs by transcarboxylase. ['H]BCCPs (4 nmol) was incubated at 30 C with 0.05 M imidazolehcl (ph 7.0) and 10 nmol of [1,3'4C]malonyl CoA (10 Ci/mol), in the presence or absence of 8.8 X 104 unit of transcarboxylase, for 15 min, in a volume of 0.1 ml. After rapid cooling of the mixture in an ice bath, '4CO2[3H]BCCP was separated from [1,3'4C]malonyl CoA on a 0.9 X 25 cm Sephadex G50 (fine) column equilibrated with 0.05 M TrisHCI (ph 9.0). Fractions of 0.5 ml were collected and aliquots were counted for both 'H and 14C. Acidlabile radioactivity was determined by adjusting aliquots to 0.02 M HCl, drying the samples, and counting. '4CRadioactivity without HCl (_), 14Cradioactivity with HCl (00), ['H]BCCP radioactivity without HCl ( 0), [3HIBCCP radioactivity with HC1 ( 0). removed by centrifugation and an additional volume of gel, equal to the first volume, was added to the supernatant solution. This was recovered by centrifugation and the two gels were pooled. The gel was washed two times with 1 ml of 0.5 M ammonium sulfate0.1 M Tris HCO (ph 7.7) per 25 mg of gel. This ammonium sulfate wash solution was added to the alumina Cy supernatant, which was saved for isolation of the transcarboxylase component (see below). E. was eluted from the gel with 0.4 M potassium phosphate, ph 7.7. For quantitative removal it was necessary to wash the gel three times with 2 ml of buffer per 25 mg of gel. The eluate, which contained E. activity, was concentrated by precipitation with ammonium sulfate at 50% saturation. The precipitate was dissolved in a minimal volume of 0.50 M imidazole 6.7). Purification of transcarboxylase component Under the conditions used, transcarboxylase is HCl (ph adsorbed to alumina Cy only to a slight extent, and most of the adsorbed enzyme is eluted with 0.5 M ammonium sulfate. The combined alumina Cy supernatantammonium sulfate wash was applied to a hydroxylapatite column (15 g of protein/liter of column volume) previously equilibrated with 0.01 M potassium phosphate (ph 7.7). The column was washed with 2 column volumes of 0.2 M potassium phosphate (ph 7.7). The transcarboxylase was eluted with 0.4 M potassium phosphate (ph 7.7) and concentrated by the addition of ammonium sulfate to 45% saturation. The resulting precipitate was dissolved in 0.05 M imidazole HCl (ph 6.7), at a protein con * Ea is a complex containing both the biotin carboxylase and BCCPL of acetyl CoA carboxylase. t Eb is the transcarboxylase component of acetyl CoA carboxylase. t One unit is defined as that amount of enzyme which catalyzes the carboxylation of 1 iamol of acetyl CoA per min under our assay conditions (3). centration of 30 mg/ml, and insoluble material was removed by centrifugation. This was applied to a 2.5 X 100 cm Sepharose 4B column previously equilibrated with 0.02 M potassium phosphate (ph 7.3)i mm EDTA0.05 M 2mercaptoethanol, and eluted with this buffer at the rate of 0.5 ml per minute. The major portion of transcarboxylase activity emerged between 330 and 400 ml. The fractions containing this activity were pooled and precipitated with ammonium sulfate at 50% saturation. The precipitate was suspended in a volume of 50% ammonium sulfate0.02 M potassium phosphate (ph 7.0) such that the protein concentration was 10 mg/ml. The precipitate was collected and successively extracted with 45, 40, 35, 30, 25, and 20% saturated ammonium sulfate in 0.02 M potassium phosphate (ph 7.0). Transcarboxylase activity was found in the 35, 30, and 25% fractions, which were pooled and brought to 50% saturation with ammonium sulfate. The resulting precipitate was dissolved in a minimal volume of 0.05 M imidazole*hcl (ph 6.7) and desalted on a Sephadex G25 column equilibrated with 0.02 M potassium phosphate (ph 7.3). The enzyme was applied to a DEAEcellulose column (10 mg of protein/ml column volume) equilibrated with 0.02 M potassium phosphate (ph 7.2). The column was washed with 2 column volumes of 0.05 M KCl 0.02 M potassium phosphate (ph 7.3), and eluted with a linear gradient of 20 column volumes of this buffer containing 0.050.12 M KCl. Fractions containing transcarboxylase activity were concentrated to 5 ml by ultrafiltration with an Amicon PM30 membrane and then brought to 50% saturation with ammonium sulfate and stored at 20 C as an ammonium sulfate suspension. RESULTS E. coli acetyl CoA carboxylase can be resolved into two components, E. and a transcarboxylase component, formerly designated as Eb (1). Fraction Ea was subsequently further dissociated to yield BCCP and biotin carboxylase. Table 1 shows the results of purification of the two components, Ea and the transcarboxylase, and demonstrates the separation of these two activities at the alumina Cy adsorption step. Further purification of transcarboxylase, which is not adsorbed

Proc. Nat. Acad. Sci. VSA 68 (1971) Transcarboxylase Component of Acetyl CoA Carboxylase 1261 TABLE 2. Malonyl CoA[W4C]acetyl CoA exchange [14C]Malonyl CoA (nmol/10 min) Complete 0.982 BCCP 0 Malonyl CoA 0 Transcarboxylase 0 Acetyl CoA (14 nmol) 1.63 The complete reaction mixture contained, in 0.09 ml: 4 nmol BCCPs, 7 or 14 nmol [214C]acetyl CoA, 13 nmol malonyl CoA, 3.5 milliunits transcarboxylase, and 53 p&mol imidazole HCl (ph 6.7). After a 10min incubation, the reaction was stopped by heating at 80'C for 5 min. Acetyl CoA was deacylated with phosphotransacetylase and the radioactivity incorporated into malonyl CoA was determined as described by Gregolin et al. (10). onto this gel, led to a 1700fold increase in specific activity; this preparation was completely free of biotin carboxylase and BCCP. However, analysis of this preparation by disc gel electrophoresis showed that it was not homogeneous at this stage, since it contained one major and several minor protein bands. The major band was identified as transcarboxylase after elution from unstained gels. Although a minimal molecular weight of 90,000 has been obtained by filtration on Sephadex G200, some preparations behave as though they have molecular weights of 180,000 or higher. Disc gel electrophoresis in the presence of sodium dodecyl sulfate (9) indicates a molecular weight of 45,00050,000, which suggests the presence of two subunits per mole of transcarboxylase. It has not been possible to ascertain whether these are identical subunits, since the preparation contains some impurities. Purified transcarboxylase is quite stable when stored as a suspension in 50% saturated ammonium sulfate. However, it loses activity in low ionic strength buffers and tends to precipitate out of solution. The availability of large quantities of pure [8H]BCCPs, labeled in the biotin prosthetic group (3), and purified transcarboxylase facilitated the direct demonstration of the transcarboxylase activity of this preparation. As shown in Fig. 1, when ['4C]malonyl CoA was incubated with transcarboxylase and [3H]BCCPs, [14C]carboxy[3H]BCCPs was formed (reverse of reaction 2), and this was readily separated from the excess radioactive substrate by filtration through Sephadex G50. Approximately 80% of the added BCCPs was carboxylated in this experiment. The 14C that cochromatographed with [3H]BCCPs was identified as 14CO2BCCPB by its acid lability, which is characteristic of this compound; ['4C]malonyl CoA is unaffected by acid. No "4Cradioactivity was found associated with [3H]BCCPs when transcarboxylase was omitted, and, of course, there was no 4Cradioactivity in the area of the void volume when BCCPs was omitted in the reaction. Since transcarboxylase catalyzes the carboxylation of BCCP by malonyl CoA, it was expected that it should catalyze a BCCPdependent exchange between [14C]acetyl CoA and malonyl CoA. Table 2 shows this exchange reaction and demonstrates the requirements for BCCP, malonyl CoA, and transcarboxylase. The results presented thus far show that purified transcarboxylase catalyzes the transcarboxylation step of the acetyl CoA carboxylase reaction. We then attempted to demonstrate an acetyl CoA and a malonyl CoA binding site on the LL. 400!..0w Z: ~~~~~~~~~~.04 200 Activity.0 10 20 FIG. 2. Binding of [214C]malonyl CoA to transcarboxylase. Transcarboxylase (0.6 mg, 2.3 units/mg) was incubated at 30 C for 15 min in 0.5 ml of imidazolehcl (ph 7.0) with [214C] malonyl CoA (20 mm, 10 Ci/mol). [214C]malonyl CoAtranscarboxylase was separated from [214C]malonyl CoA as described in Fig. 1, except the column was equilibrated with 0.05 M imidazolehcl (ph 6.7) and fractions of 0.4 ml were collected. Aliquots were counted and assayed for transcarboxylase activity. [14CJMalonyl CoA radioactivity with transcarboxylase present (14); [14C]malonyl CoA radioactivity with transcarboxylase absent (); transcarboxylase activity, designated as Eb activity (0C). protein. These experiments were complicated by the properties of transcarboxylase discussed above; i.e., it is very unstable in low ionic strength and tends to precipitate out of solution. Thus, when transcarboxylase was mixed with a labeled acyl CoA and then filtered through Sephadex to separate the enzymebound substrate from the starting substrate, activity was largely lost during the filtration procedure. However, some activity always survived, and labeled substrate was found associated with the enzyme peak. This is illustrated in Fig. 2, which demonstrates the binding of [2'4C]malonyl CoA by transcarboxylase. A mixture of enzyme and [14C] malonyl CoA was filtered through Sephadex G50; it is apparent that a radioactive peak coincided with enzyme activity in the void volume. In this experiment, 20% of the transcarboxylase activity was found in the peak. The amount of radioactivity associated with transcarboxylase was dependent upon the concentration of ['4C]malonyl CoA and enzyme, as shown in Table 3. Addition of unlabeled acetyl CoA decreased the amount of ['4C]malonyl CoA bound to transcarboxylase, which suggests that these two acyl CoA derivatives are bound at a common site. Numerous attempts were made to stabilize the transcarboxylase so that the acyl CoA site could be better demonstrated. However, variations in ph and ionic strength, within the limits permitted by enzyme activity, did not improve the results shown in Fig. 2. When higher concentrations of enzyme were used, the protein precipitated on the Sephadex column. This is illustrated in the experiments of Fig. 3, where 5.8 mg of transcarboxylase was incubated with [14C]acetyl CoA and then filtered through a Sephadex G50 column. When the

1262 Biochemistry: Alberts et al. Proc. Nat. Acad. Sci. USA 68 (1971) FIG. 3. Binding of ["4C]acetyl CoA to transcarboxylase. Transcarboxylase (5.8 mg, 3.4 units/mg) was incubated at 30'C for 15 min, in 0.5 ml, with [2'4C]acetyl CoA (1.4 X 104 M, 20 Ci/mol). [2'4C]Acetyl CoAtranscarboxylase was separated from [214C]acetyl CoA as described in Fig. 1, except that the column was equilibrated with 0.05 M imidazole HCI (ph 6.7) and fractions of 0.28 ml were collected. Aliquots were counted and assayed for transcarboxylase activity. 14CRadioactivity with transcarboxylase present (); 14CRadioactivity with transcarboxylase absent (0); transcarboxylase activity (OO). reaction mixture entered the gel, a white band appeared immediately, slowly moved down the column, and emerged as a turbid suspension. As noted in Fig. 3, only trace amounts of enzyme activity and radioactivity were present in the void volume (arrow) where they are normally found. Transcarboxylase activity was found in the turbid fractions (2530) that preceded the salt peak; associated with the enzyme activity was a shoulder of [14C]acetyl CoA radioactivity. This shoulder did not occur when transcarboxylase was omitted. Thus, association of acetyl CoA with transcarboxylase was demonstrated even under conditions in which protein was obviously precipitating out of solution. The enzyme activity in fractions 2530 was rapidly lost and could not be recovered by suspension of the insoluble protein in buffers of high ionic strength. TABLE 3. Binding of malonyl CoA to transcarboxylase [214C] Malonyl [214C] Malonyl Acetyl Trans CoA CoA CoA carboxylase transcarboxylase (M) (M) (mg) (nmol) 4.0 X 105 0.6 0.0292 1.2 X 104 0.6 0.0775 2.0 X 104 0.6 0.0918 4.0 X 104 0.6 0.1305 2.0 X 104 2.0 X 104 0.6 0.0545 2.0 X 104 0.2 0.0394 [214C] Malonyl CoAtranscarboxylase was prepared as decribed in Fig. 2, except that the quantities of [214C] malonyl CoA, acetyl CoA, and transcarboxylase indicated above were used. 10 Eb (Units x103) FIG. 4. Effect of transcarboxylase on the stability of C02 BCCP. 14CO2BCCP (0.13 nmol), prepared as described (1, 3), was incubated at 330C in 0.32 ml with the quantity of transcarboxylase (Eb) indicated and 0.05 M imidazole HCl (ph 7.5). 50,A aliquots were removed at various times and the amount of "4CO2BCCP remaining was determined by the addition of 0.03 umol of acetyl CoA and 0.02 unit of transcarboxylase; the formation of malonyl CoA was assayed (1). T1/, was calculated from a semilog plot of the percentage of 14CO2BCCP remaining at different times. Thus, although these experiments demonstrate binding site(s) for both malonyl CoA and acetyl CoA, the extreme lability of transcarboxylase did not permit further characterization of the site(s). Evidence was presented earlier that transcarboxylase interacts with BCCP (1). This evidence was the finding that the reaction of C02BCCP with avidin is greatly enhanced in the presence of transcarboxylase. Transcarboxylase also influences the rate of C02BCCP decarboxylation. This is illustrated in Fig. 4, where the T1/, of 14CO2BCCP was studied both in the absence and in the presence of increasing concentrations of enzyme. [14C]CarboxyBCCP is very unstable at ph 7.5 and 33 C, exhibiting a T1/, of 27.5 min. The T1/, was, however, drastically decreased in the presence of transcarboxylase; with 0.16 unit of enzyme present, the Ti/, dropped to 6 min. Thus, it is apparent that in the absence of the acceptor, acetyl CoA, the transcarboxylase component catalyzes the decarboxylation of C02BCCP (reaction 4). DISCUSSION We earlier suggested that a protein fraction from E. coli, which was designated Eb, catalyzes the transcarboxylation step of the acetyl CoA carboxylase reaction (13). This suggestion was based upon the findings that this component was required for the transfer of the carboxyl group from CO2BCCP to acetyl CoA, that it catalyzed the formation of C02BCCP from malonyl CoA and BCCP, and that it was not required in the MnATPdependent carboxylation of BCCP. However, in all these studies, the participation of biotin carboxylase in the transcarboxylation reaction could not be ruled out since the biotin carboxylase was present in the enzyme preparations as a contaminant. The availability of pure BCCP and highly purified transcarboxylase has now permitted an unequivocal demonstration that transcarboxylase catalyzes reaction 2.

Proc. Nat. Acad. Sci. USA 68 (1971) Evidence supporting this proposal includes the demonstration that transcarboxylase (a) catalyzes the carboxylation of BCCP by malonyl CoA, (b) catalyzes the BCCPdependent exchange between ['4C]acetyl CoA and malonyl CoA, (c) binds labeled acetyl CoA and malonyl CoA, and (d) catalyzes the decarboxylation of C02BCCP. Since biotin carboxylase was not present in the experiments described above, it is clear that it is not required in these reactions. Thus, biotin carboxylase participates directly only in reaction 1 and transcarboxylase participates directly solely in reaction 2; only BCCP is involved in both partial reactions of acetyl CoA carboxylase. These experiments, however, do not rule out the possibility that the various subunits might interact when they are associated in a complex in such a way that the complex would exhibit greater activity than is manifested by the individual subunits. Although an intact complex containing biotin carboxylase, BCCP, and transcarboxylase has not yet been isolated, we have observed on several occasions that these three proteins have cochromatographed on Sepharose after the alumina C'y step in the purification procedure; this observation suggests that such a complex probably exists (Alberts, A. W., unpublished observations). From the experiments presented, we conclude that transcarboxylase contains site(s) for the acyl group and for biotin, the covalently bound prosthetic group of BCCP. Experiments to determine whether the transcarboxylase covalently binds the acyl group of acetyl and malonyl CoA, as reported by Heinstein and Stumpf (11) for wheat germ acetyl CoA carboxylase, or whether it binds the acyl CoA molecule, have been precluded by the instability of the transcarboxylase noted above. Information as to the nature of the interaction of Transcarboxylase Component of Acetyl CoA Carboxylase 1263 transcarboxylase with BCCP may be forthcoming when sequence studies and xray crystallographic studies (presently in progress) in other laboratories on BCCP are completed. NOTE ADDED IN PROOF While this manuscript was in preparation, a report appeared [Guchhait, R. B., J. Moss, W. Sokolski, and M. D. Lane, Proc. Nat. Acad. Sci. USA, 68, 653 (1971)] demonstrating that a preparation of the transcarboxylase component catalyzed the transfer of the carboxyl group of malonyl CoA to (+) biotin, which confirms the role of this protein in a model system. The expert technical assistance of Miss Janice Maul is gratefully acknowledged. This work was supported by grants NIH RO1HE10406 and NSF GB5142X. 1. Alberts, A. W., and P. R. Vagelos, Proc. Nat. Acad. Sci. USA, 59, 561 (1968). 2. Alberts, A. W., A. M. Nervi, and P. R. Vagelos, Proc. Nat. Acad. Sci. USA, 63, 1319 (1969). 3. Nervi, A. M., A. W. Alberts, and P. R. Vagelos, Arch. Biochem. Biophys., in press. 4. Fall, R. R., A. M. Nervi, A. W. Alberts, and P. R. Vagelos, Proc. Nat. Acad. Sci. USA, in press. 5. Dimroth, P., R. B. Guchhait, E. Stoll, and M. D. Lane, Proc. Nat. Acad. Sci. USA, 67, 1353 (1970). 6. Simon, E. J., and D. Shemin, J. Amer. Chem. Soc., 75, 3520 (1953). 7. Trams, E. G., and R. 0. Brady, J. Amer. Chem. Soc., 82, 2972 (1960). 8. Davis, B. J., Ann. N.Y. Acad. Sci., 121, 404 (1964). 9. Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 (1969). 10. Gregolin, C., E. Ryder, and M. D. Lane, J. Biol. Chem., 243, 4227 (1968). 11. Heinstein, P. F., and P. K. Stumpf, J. Biol. Chem., 244, 5374 (1969).