Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp. 653-657, March 1971

The Carboxyl Component of Acetyl CoA Carboxylase: Structural Evidence for Intersubunit Translocation of the 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 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 , 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 of for Eb, catalyzes BC- and CCP-independent carboxyl transfer acetyl CoA. The transcarboxylation , after stabil- ization by , 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 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 . 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 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. from [2-'4C]malonyl CoA or of ['4C]carboxybiotin from [3- t Pooled malonyl CoA decarboxylase (MCD)-containing 14C]malonyl CoA is determined by the malonyl CoA decar- fractions from phosphocellulose chromatography, free of the boxylase assay procedure modified to include 10 mM free d- biotin-containing carboxyl-carrier-protein (Fig. 1B). 28 jug was biotin as carboxyl acceptor. The '4C-labeled fragments ([2- used to measure acetyl CoA carboxylase activity. 14C]acetic acid or [14C]C02) derived from either radioactive t- - -, not determined. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Intersubunit Translocation of Biotin 655

biotin carboxylase and carboxyl-carrier-protein. It is evident (Fig. 1A and B) that the elution patterns for malonyl CoA 550 decarboxylase and the restoration of acetyl CoA carboxylase b _ _=a-- .:-none- activities are superimposable, suggesting that a single enzy- X ~~-_ ~>enz matic component is responsible for both activities. It is also E 500 enz evident (Table 1) that acetyl CoA carboxylation catalyzed by - d-biotin the pooled phosphocellulose column fractions is absolutely dependent upon the addition of the biotin carboxylase- 450 carboxyl-carrier-protein preparation. The phosphocellulose- purified enzyme obtained from an E. coli biotin auxotroph u (Strain SA 283) grown on [2-14C]biotin of high specific activ- c 400 ity (57 Ci/mol) was free of radioactivity, indicating that Eb Stable tc gassing with CC was completely resolved from the biotin-containing carboxyl- 02 _enz + carrier-protein. D 350 _Stable toI) -ocid ot_950 I d-biotin Since this essential component of the acetyl CoA carboxyl- 0

ase system also appears to decarboxylate malonyl CoA, it is w 1 15 must possess a site for CoA. w 0,I evident that it binding malonyl L)- 5 10 15 This, and the fact that the abortive reaction is chemically re- MINUTES INCUBATION lated to the reverse of Reaction 2, suggested that this com- FIG. 3. Kinetics of formation of an acid-labile carboxyl trans- ponent may function as a carboxyl transferase. fer product. The carboxyl transferase assay reaction mixture Eb-catalyzed earboxyl transfer from malonyl CoA to free contained [3-'4C]malonyl CoA (6400 cpm per nmol) and 15 jg of biotin phosphocellulose-purified Eb-MCD (Fig. iB; carboxyl trans- ferase specific activity, 98 milliunits per mg). Acid-stable 14C was In addition to malonyl CoA decarboxylation, the enzyme also determined according to the malonyl CoA decarboxylase assay catalyzes carboxyl transfer from malonyl CoA to free d-biotin, procedure, and 14C stable to gassing with C02 was determined as which serves as a model for the reverse of partial Reaction 2 of described in the legend to Table 2. the overall process. This model reaction can be followed in- directly, by determining the rate of d-biotin-dependent forma- tion of ['4C]acetyl CoA from [2-14C]malonyl CoA, or di- the carboxyl transferase is highly specific in that l-biotin does rectly, by measuring carboxybiotin formation. As illustrated not enhance the rate of acetyl CoA formation (Fig. 2) and is in Fig. 2, the rate of acetyl CoA formation is enhanced about completely inactive as an acceptor at concentrations ranging 12-fold by 10 mM d-biotin and is proportional to enzyme con- from 2 to 20 mM. These results show unequivocally that this centration. In the presence of d-biotin and ['4C]carboxyl- enzyme, which is an essential component of the E. coli acetyl labeled malonyl CoA, an acid-labile compound is formed that CoA carboxylase system, has binding sites for both malonyl has stability properties characteristic of N-carboxybiotin, CoA and d-biotin, the model prosthetic group, and that it i.e., it is stable to gassing with CO2 (see Fig. 3). In addition, catalyzes carboxyl transfer between the two compounds. The Km values for malonyl CoA and d-biotin in the car- boxyl-transferase reaction are 0.10 mM and 3.5 mM, respec- tively. Since the Km for free biotin in this reaction is nearly two orders of magnitude lower than that in the biotin carbox- 25 ylase-catalyzed ATP-dependent carboxylation of free biotin, 24ug enz+d-biotin the carboxyl transferase component appears to have a much E higher affinity for biotin than does the biotin carboxylase 20 c: component. Interestingly, 2-imidazolidone (100 -mM), an w analogue of the functional ureido ring of biotin, activates = 100 CoA 0 15 (KA mM) [3-14C]malonyl decarboxylation al- UL though it does not appear to serve as a carboxyl acceptor. 0 /12Eg enz +d-biotin The binding of imidazolidone, presumably at the biotin site

-J 10 may promote a conformational change at the malonyl CoA site that enhances the abortive decarboxylation. cILi 5 Identification of 1'-N-carboxy-d-biotin as carboxyl transfer product / / _ = 24tPg enz± I-biot in The acid-labile product formed by incubating [3-14C]malonyl tP- 11, 12j2g enz 5 10 15 CoA and d-biotin with the carboxyl transferase component MINUTES INCUBATION was characterized after stabilization with diazomethane. As indicated in Table 2 (A), after a 15-min incubation of [3- FIG. 2. Effect of free d- and l-biotin on the rate of Eb-de- 14C]malonyl CoA and d-biotin with carboxyl transferase, pendent acetyl CoA formation from malonyl CoA. The rate of about of the group of was formation of [2-'4C]acetyl CoA from [2-14C]malonyl CoA was 70% [3-14C] carboxyl malonyl CoA determined by the carboxyl transferase assay procedure using converted to a form stable to gassing with C02, but labile in phosphocellulose-purified Eb-MCD (Fig. 1B; carboxyl transferase acid. 30% of the [14C]CO2 was presumably lost by enzymatic specific activity, 100 milliunits per mg). Free d- and l-biotin were decarboxylation of malonyl CoA and by nonenzymatic de- added at 10 mM and 16 mM, respectively. carboxylation of N-carboxybiotin (ti/, about 60 min, unpub- Downloaded by guest on September 28, 2021 656 Biochemistry: Guchhait et al Proc. Nat. Acad. Sci. USA 68 (1971)

lished observations). After methylation, about 65% of the the chromatogram was due to [14C]dimethylmaloliate, which radioactive product was recovered in the form of an acid- arose from a 13% [14C]malonate contaminant in the enzy- stable compound that behaved on paper chromatography matically-synthesized malonyl CoA preparation and resulted (Fig. 4) as authentic 1'N-carboxy-d-biotin dimethyl ester. from nonenzymatic deacylation. The identification of the The small amount of radioactivity remaining at the origin of methylated '40-labeled carboxyl transfer product as 1'-N- carboxy-d-biotim dimethyl ester was further verified by carrier recrystallization to constant specific activity with the authen- TABLE 2. Characterization of the carboxyl transfer product tic, chemically-synthesized, compound (Table 2B). A. Stability and recovery of "4C-labeled carboxyl transfer DISCUSSION product: E. coli acetyl CoA carboxylase is comprised of three essential Radioactivity protein components (3-7): a) carboxyl-carrier-protein (CCP), recovered* which contains the covalently-bound biotin prosthetic group, Step (cpm) (b) biotin carboxylase which catalyzes the ATP-dependent 1. Before incubation carboxylation of CCP-bound or free biotin, and (c) an addi- [3-14C] malonyl CoA-acid stable 1,010,000 tional protein, Eb, which is believed to have a role in carboxyl 2. After incubation transfer from the carboxylated prosthetic group to acetyl "[14C] carboxybiotin"-acid labile, stable to CoA, although it was not possible, until now, to assign a func- gassing with C02 670,000t tion to this component. In addition to a potential catalytic 3. After incubation and methylation role for Eb, other possibilities existed, including that of a " [14C] carboxybiotin (acid stable4 430,000 "specifier" analogous to the mode of action of lactalbumin dimethyl ester" cocrystallizable in the lactose synthetase system 14). The present with carrier (13, investi- 440,000 gation clearly defines the catalytic role of this component. Eb catalyzes carboxyl transfer from malonyl CoA to free d- B. Carrier crystallization of methylated "C-labeled product biotin, to form acetyl CoA and N-carboxybiotin, a model for with authentic 1'-N-carboxy-d-biotin dimethyl ester: the reverse of the second half-reaction (Reaction 2) in the Melting Specific carboxylation of acetyl CoA. In addition, it carries out a slow, point Recovery activity abortive, biotin-independent decarboxylation of malonyl Crystallization (0C) (mg) (cpm/mg) CoA, which is a property shared by several other acyl CoA 1st (MeOH-water) 130-1 143 1750 carboxylases, namely avian liver acetyl CoA carboxylase and 2nd (MeOH-water) 131-2 96.4 1720 pig-heart propionyl CoA carboxylase. These results show that 3rd (toluene-pet. ether) 131-2 93.3 1760 Eb possesses binding sites for d-biotin and for malonyl CoA. 4th (toluene-pet. ether) 131-2 91.0 1750 Furthermore, preliminary experiments in our laboratory (S. E. Polakis and P. Dimroth) show that Eb catalyzes a carboxyl- * Corrected for 13% ['4C]malonate present in [3-14C]malonyl carrier-protein-dependent malonyl CoA-[2-14C]acetyl CoA CoA substrate. exchange, demonstrating the presence of an acetyl CoA site t The radioactivity labile to gassing with CO2 after incubation and completing the requisite complement of substrate and is attributed to ['4C] CO2 arising from the enzymatic decarboxyl- prosthetic group binding sites expected of a carboxyl trans- ation of malonyl CoA and nonenzymatic decarboxylation of ferase. carboxybiotin. Although neither enzymatic component, biotin $ Identified as l'-N-carboxy-d-biotin dimethyl ester by paper carboxylase chromatography (see Fig. 4). nor carboxyl transferase, contains covalently-bound biotin, The reaction mixture (final volume, 0.5 ml), which contained 100 mM imidazole HCl buffer, pH 6.7; 0.34 mM [3-14C] malonyl CoA (6.4 X 106 cpm per pmol); 10 mM d-biotin; 0.3 mg of bovine serum albumin; and 0.3 mg of phosphocellulose-purified Eb-malonyl CoA decarboxylase (Fig. 1B, carboxyl transferase specific activity, 100 milliunits per mg), was incubated at 30°C for 15 min. A control without d-biotin was run in parallel. 14C stable to with was activity gassing CO2 determined after trans- DIMETHYL d-BIOTIN I-N-CARBOXY- 3'-N-CARBOXY-. ferring a 0.05-mil aliquot to 1.45 ml of 67 mM triethanolamine HCl MALONATE METHYL d-BIOTIN DIMETHYL ESTER buffer, pH 8, at 0°C and bubbling a stream of C02 through the ESTER solution for 20 min. Acid-stable 14C was determined by transferring FIG. 4. Chromatographic identification of the methylated a 0.05-ml aliquot to a scintillation counting vial containing either carboxyl-transfer product. An aliquot of the methylated product 0.1 ml of 6 N HCl (Steps 1 and 2) or 0.1 ml of 6 N acetic acid (Step of transcarboxylation from [3-14C]malonyl CoA to free d-biotin 3) and then taking the acidified solutions to dryness. 4 ml of (Step 3, Table 2) was subjected to descending paper chromatog- methanol at 0°C was added immediately after incubation to the raphy at 40C, using the solvent system of Knappe et al. (12); remainder (0.4 ml) of the reaction mixture, followed by sufficient authentic reference compounds were cochromatographed, as 1 M diazomethane in ether to give a persistent yellow color. Ali- well as run separately. Developed chromatograms were scanned quots were taken for the paper chromatographic identification of for radioactivity (shown above) and sprayed with 0.2% KMnO4 the "4C-labeled product. After the addition of 200 mg of chemi- to locate the reference compounds. 1'-N- and 3'-N-carboxy-d- cally-synthesized carrier 1'-N-carboxy-d-biotin dimethyl ester biotin dimethyl esters were synthesized from d-biotin methyl ester (12), the derivative was recrystallized 2 times from methanol- and methylehloroformate (12); dimethylmalonate and d-biotin water 95:5 and 2 times from toluene-petroleum ether; the specific methyl ester were prepared by methylation of the free acids with activity and melting point of the product were determined. diazomethane. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Intersubunit Translocation of Biotin 657 It is apparent that the bicyclic ring of the biotinyl pros- thetic group of the carboxyl-carrier-protein must oscillate be- tween two catalytic sites, i.e., biotin carboxylating and car- boxyl transferring, which are housed on different subunits (see A Fig. 5). The present investigation provides the first direct structural evidence for intersubunit translocation of the carboxylated biotinyl group and is supported by kinetic evidence for "ping-pong" mechanisms obtained with two other biotin-dependent enzymes, i.e, bacterial methylmalonyl (15, 16) and liver acetyl C'B2 CoA: pyruvate transcarboxylase CoA carboxylase (17, 18). This work was supported by research grants from the National Institutes of Health, USPHS (AM-14574 and AM-14575), and B. the American Heart Association, Inc. J. M. was supported by USPHS Medical Scientists Training Grant 5TO5 GM-01668 (New York University School of Medicine). 1. Kaziro, Y., and S. Ochoa, Advan. Enzymol., 26, 283 (1964). 2. Moss, J., and M. D. Lane, Advan. Enzymol., 34, in press (1971). 3. Alberts, A. W., and P. R. Vagelos, Proc. Nat. Acad. Sci., USA, 59, 561 (1968). yN-Co A 4. Alberts, A. W., A. M. Nervi, and P. R. Vagelos, Proc. Nat. Acad. Sci., USA, 63, 1319 (1969). for intersubunit translocation of 5. Dimroth, P., R. B. Guchhait, E. Stoll, and M. D. Lane, FIG. 5. Hypothetical scheme Proc. Nat. Acad. Sci., USA, 67, 1353 (1970). the carboxylated biotin prosthetic group of acetyl CoA carbox- 6. Dimroth, P., R. B. Guchhait, and M. D. Lane, Z. Physiol. ylase. to carboxylase, CCP to carboxyl-carrier- BC refers biotin Chem., in press. protein, and CT to carboxyl transferase. 7. Nervi, A. M., and A. W. Alberts, Fed. Proc., 29, 333 (1970). 8. Trams, E., and R. 0. Brady, J. Amer. Chem. Soc., 82, 2972 both catalyze model reactions with free biotin that account for (1960). their respective catalytic functions in the half-reactions (Reac- 9. Gregolin, C., E. Ryder, and M. D. Lane, J. Biol. Chem., 243, 4227 (1968). tions 1 and 2) of the overall process. It is evident that both the 10. Simon, E. J., and D. Shemin, J. Amer. Chem. Soc., 75, 2320 biotin carboxylase and carboxyl transferase components con- (1953). tain distinct biotin binding sites. Furthermore, these sites 11. Warburg, O., and W. Christian, Biochem. Z., 310, 384 are highly specific since free l-biotin cannot replace free d- (1942). biotin in either model reaction (Fig. 2 and ref. 5). On the 12. Knappe, J., E. Ringelmann, and F. Lynen, Biochem. Z., 335, other hand, the biotin-binding sites of the two enzymes differ 168 (1961). markedly with respect to their apparent affinities for free d- 13. Brew, K., T. C. Vanaman, and R. L. Hill, Proc. Nat. Acad. biotin; biotin carboxylase and carboxyl transferase have Km Sci., USA, 59, 491 (1968). values for free biotin of 170 mM (5) and 3 mM, respectively. 14. Fitzgerald, D. K., U. Brodbeck, I. Kiyosawa, R. Mawal, and K. E. J. Biol. Chem., 245, 2103 (1970). Interestingly, both enzyme components have far lower affini- B. Colvin, Ebner, ties for free carboxybiotin. Enzymatically-synthesized, free, 15. Northrop, D. B., J. Biol. Chem., 244, 5808 (1969). [I4C]carboxy-d-biotin preparations (containing 10-15 mM 16. Northrop, D. B., and H. G. Wood, J. Biol. Chem., 244, 5820 and approximately equimolar free (1969). free [14C]carboxy-d-biotin 17. Hashimoto, T., N. Iritani, S. Nakanishi, and S. Numa, d-biotin) were not decarboxylated by biotin carboxylase in Proc. Japanese Conf. on Biochem. Meetings, Ikaho, the presence of ADP, Pi, Mg2+, hexokinase, and glucose, nor July, 1970, p. 21. did they undergo carboxyl transfer to acetyl CoA in the pres- 18. Numa, S., S. Nakanishi, T. Hashimoto, N. Iritani and T. ence of carboxyl transferase. Okazaki, Vitam. Horm., in press (1970). Downloaded by guest on September 28, 2021