Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp. 653-657, March 1971 The Carboxyl Transferase Component of Acetyl CoA Carboxylase: Structural Evidence for Intersubunit Translocation of the Biotin Prosthetic Group RAS B. GUCHHAIT, JOEL MOSS, WALTER SOKOLSKI, AND M. DANIEL LANE Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by Albert L. Lehninger, January 11, 1971 ABSTRACT An essential protein component of acetyl berts, et al. (3), which is free of biotin and appears to function CoA carboxylase, isolated and extensively purified from in the second half-reaction. The precise role of Eb, whether cell-free extracts of Escherichia coli, has been identified as malonyl CoA:d-biotin carboxyl transferase. This enzyme, catalytic, structural, or otherwise, has remained obscure. which does not contain covalently-bound biotin, catalyzes The present investigation reveals that a protein isolated carboxyl transfer from malonyl CoA to free d-biotin, a from E. coli, having characteristics similar to those reported model reaction for the second step in the carboxylation of for Eb, catalyzes BC- and CCP-independent carboxyl transfer acetyl CoA. The transcarboxylation product, after stabil- ization by methylation, was identified as 1'-N-carboxy-d- from malonyl CoA to free d-biotin to form free carboxybiotin. biotin dimethyl ester. These results indicate the presence This malonyl CoA: d-biotin carboxyl transferase, which has of a biotin site on the carboxyl transferase, distinct from been extensively purified, is devoid of biotin and is required that on the biotin carboxylase, which carries out the first in combination with BC and CCP for acetyl CoA carboxyla- step in the overall process. In addition, the carboxyl tion. transferase catalyzes a slower abortive decarboxylation of malonyl CoA, thus indicating that carboxyl abstraction and protonation do not require the participation of EXPERIMENTAL PROCEDURE biotin. E. coli B cells (grown to '/4 log phase) grown on enriched It is now evident that the half-reactions of acetyl CoA carboxylation are catalyzed by biotin carboxylase and medium were purchased from Grain Processing Corp., carboxyl transferase. Both components are devoid of Muscatine, Iowa. E. coli SA 283, a biotin auxotroph, was biotin and have specific binding sites for free d-biotin, as grown in the presence of [2'-'4C] d-biotin as described (5). well as for their respective substrates; hence, the acetyl Biotin carboxylase assays, materials, and other procedures not CoA carboxylation mechanism must involve intetsubunit described herein were as reported (5, 6). CoA translocation of the carboxylated biotinyl group, which is Acetyl carboxyl- bound covalently to carboxyl-carrier-protein, a non- ation was determined at 30°C by measuring ['4C]bicarbonate catalytic polypeptide. incorporation into malonyl CoA under assay conditions simi- lar to those of Alberts and Vagelos (3). [2-'4C]malonyl CoA It is now well established that the reactions catalyzed by was chemically synthesized by the method of Trams and acetyl CoA carboxylase and other biotin-dependent carbox- Brady (8) and [3-'4C]malonyl CoA was enzymatically syn- ylases (1, 2) proceed via the minimal 2-step reaction sequence thesized according to Gregolin, et al. (9); both labeled thio- shown below: esters were purified as described (9). Acetyl CoA was prepared Me2 + by the method of Simon and Shemin (10). Protein concentra- Enz-biotin + HCO3- + ATP = Enz-biotin-CO2, + tion was determined spectrophotometrically (11). ADP + Pi (1) Steps in the preparation of carboxyl transferase Enz-biotin-CO2- + Acceptor =± Enz-biotin + Cell-free extracts of E. coli are prepared in 0.1 M potassium Carboxylated phosphate buffer, pH 7, using a Manton-Gaulin submicron Acceptor (2) dispersor. The enzyme is purified by fractionation with am- (e.g., acetyl CoA) (e.g., malonyl CoA) monium sulfate (between 25 and 42% saturation), adsorption Unlike the carboxylases from higher organisms, which retain on and elution from calcium phosphate gel, and ion-exchange their structural chromatography on DEAE-cellulose and phosphocellulose. integrity during purification (2), Escherichia This coli acetyl CoA carboxylase is readily resolved into three es- procedure, which will be reported in detail elsewhere, sential protein components (3, 4): (a) biotin carboxylase results in preparations that are at least 200-fold purified (BC), which catalyzes the ATP- and divalent cation-depen- and have a specific activity in the carboxyl transferase assay dent carboxylation of biotin (4-6) and presumably participates of approximately 100 milliunits per mg of protein. in the first half-reaction [Reaction (1)], (b) carboxyl-carrier- Malonyl CoA decarboxylase assay protein (CCP), a polypeptide of about 9000 daltons, which contains a covalently-bound biotin prosthetic group (7), The rate of malonyl CoA decarboxylation is determined in a and (c) a third protein component, referred to as Eb by Al- reaction mixture (0.5 ml, pH 6.7) containing 100 mM imid- azole HCI buffer, 85 ,uM [2-14C]- or [3-14C]malonyl CoA Abbreviations: BC, biotin carboxylase; CCP, carboxyl-carrier- (4-6 X 103 cpm per nmol), 0.3 mg of bovine serum albumin, protein; MCD, malonyl CoA decarboxylase. and up to 10 milliunits of carboxyl transferase. At 5, 10, 15, 653 Downloaded by guest on September 28, 2021 654 Biochemistry: Guchhait et al. Proc. Nat. Acad. Sci. USA 68 (1971) product during the work-up subsequent to the enzymatic reaction are volatilized; this procedure leaves behind the re- sidual acid-stable '4C from unused substrate. The rate of car- boxyl transfer is equal to the difference between the rate of dis- appearance of acid-stable 14C in the presence and absence of free d-biotin. Linear transfer rates are obtained for 10 min with up to 2 milliunits of carboxyl transferase. One unit of carboxyl transferase catalyzes the formation of 1 Mimol of free carboxybiotin per min from malonyl CoA and free d-biotin under these conditions. RESULTS Isolation of Eb, an essential component of the carboxylase system that possesses malonyl CoA decarboxylase activity Investigations in this laboratory (J. Moss, unpublished ob- 200 ELUATE VOLUME ml servations) have shown that several biotin-dependent carbox- ylases catalyze a slow, avidin-insensitive, decarboxylation of FIG. 1. Cochromatography of Eb and malonyl CoA decar- their respective carboxylated acceptor substrates, e.g., mal- boxylase (MCD). (A) Calcium phosphate gel-purified enzyme onyl CoA decarboxylation by liver acetyl CoA carboxylase. (1.46 g of protein, see preparation of carboxyl transferase in Hence, these enzymes can labilize the a-carboxyl group of Experimental Procedure) in 10 mM potassium phosphate buffer, their carboxylated acceptors (e.g., malonyl CoA) and insert a pH 7.0, containing 1 mM EDTA and 5 mM ,-mercaptoethanol proton without the participation of the biotin prosthetic was applied to a 4.5 X 50 cm DEAE-cellulose column and group. component E. eluted with a 2-liter linear phosphate gradient (50-400 mM, pH 7) Our suspicion that the Eb of the coli also containing EDTA and fl-mercaptoethanol. The eluted frac- acetyl CoA carboxylase system might catalyze this abortive tions were assayed for MCD activity and for the ability to restore reaction proved correct and provided a means to assay and acetyl CoA carboxylase [in the presence of 0.96 mg of a combined follow this component during fractionation. After partial biotin carboxylase-carboxyl-carrier-protein preparation, calcium resolution from the biotin carboxylase and carboxyl-carrier- phosphate gel-purified enzyme from Step 3 of the biotin car- protein components by ammonium sulfate and calcium phos- boxylase purification procedure (5)]. The enzymatically-active phate gel fractionation, the malonyl CoA decarboxylase activ- fractions were pooled, and the protein was precipitated with 60%- ity was purified further by ion-exchange chromatography on saturated ammonium sulfate. (B) After dialysis against 25 mM DEAE-cellulose (Fig. 1A) and phosphocellulose (Fig. 1B). potassium phosphate buffer, pH 7, containing 1 mM EDTA and ,B-mercaptoethanol, half of the protein (25 mg) recovered In order to determine whether the enzyme having malonyl an from A was applied to a 1.5 X 30 cm column of phosphocellulose. CoA decarboxylase activity is essential component of the Elution was with a 500-ml phosphate gradient (25-300 mM, pH 7) acetyl CoA carboxylase system, the column fractions were containing EDTA and f3-mercaptoethanol. The eluted fractions assayed both for biotin-independent malonyl CoA decarbox- were assayed and the active fractions precipitated as in A. ylase activity and for their ability to restore acetyl CoA carboxylase activity to an enzyme preparation containing and 20 min of incubation at 300C, 0.1-ml aliquots are trans- TABLE 1. Reconstitution ofacetyl CoA carboxylase activity ferred to scintillation vials containing 0.1 ml of 6 N HCL. The acidified solutions are taken to dryness at 95°C; water and scintillator are added, and the residual acid-stable 14C is Specific enzyme activity Acetyl- determined. The [14C]C02 or [2-14C]acetic acid generated are CoA volatile under these conditions, whereas [2-14C]malonyl CoA Malonyl-CoA carboxyl- is not. Biotin decar- Carboxyl ated/5 carboxylase boxylase transferase min Carboxyl transferase assay Enzyme (munits/mg) (munits/mg) (munits/mg) (nmol) Malonyl CoA: d-biotin carboxyl transferase (CT) catalyzes BC-CCP* 2.9 0.05 0.2 3.5 transcarboxylation from malonyl CoA to free d-biotin (Reac- MCDt 0.0 8 96 0.0 tion 3), a model reaction for the reverse of the second step BC-CCP* (Reaction 2). + MCDt -----40 Malonyl CoA + d-biotin > * Combined biotin carboxylase (BC)- and carboxyl-carrier- CT protein (CCP)- containing enzyme preparation; calcium phos- acetyl CoA + carboxy-d-biotin (3) phate gel purified enzyme from Step 3 of the biotin carboxylase purification procedure (5). 0.96 mg was used to measure acetyl The (free) biotin-dependent formation of [2-14C]acetyl CoA CoA carboxylase activity.