Studies of the Acetyl Coenzyme A Synthetase Reaction

THE JOURNAL OF BOLOQCAL CHEMSTRY Vol. 240, No. 11, November 1965 Printed in U.S.A. Studies of the Acetyl Coenzyme A Synthetase Reaction. EVDENCE OF A DOUBLE REQUREMENT FOR DVALENT CATONS* LESLE T. WEBSTER, JR.~ WTH THE TECHNCAL ASSSTANCE OF NANCY CARTER From the Departments of Medicine and Biochemistry, Western Reserve University, School of Medicine, Cleveland, Ohio 44106 (Received for publication, April 28, 1964) n contrast to their function in reactions involving a cleavage of adenosine triphosphate into adenosine diphosphate and inorganic phosphate (l), little is known about the role of divalent cations in certain synthetase reactions characterized by cleavage of adenosine triphosphate to adenosine monophosphate and pyrophosphate with the formation of enzyme-bound acyladenylates as intermediates. Examples of the latt,er type of reaction include the activation of many fatty and amino acids, luciferin, cholate, lipoate, and pantoate (2). Therefore, divalent metal ion requirements were determined for the acetyl coenzyme A synthetase reaction (Equation 1) in which there is reversible formation of acetyl coenzyme A, adenosine monophosphate, and pyrophosphate from acetate, adenosine triphosphate, and coenzyme A. Acetate + ATP + CoA e acetyl-coa + AMP + PPi (1) Acetate + ATP + E = E(acetyladenylate) + PPi (2) E(acetyladenylate) + CoAe acetyl-coa + AMP + E (3) The acetyl-coa synthetase system was chosen for two principal reasons. First, a well characterized crystalline enzyme of known molecular weight can be prepared from bovine heart mitochondria (3). Second, in addition to the rate of the over-all reaction, two partial reactions can be evaluated under equilibrium conditions; both of these involve the intermediate formation of enzyme-bound acetyladenylate. n the first partial reaction the intermediate is formed from acetate and adenosine triphosphate (Equation 2) and in the second, from acetyl coenzyme A and adenosine monophosphate (Equation 3) (4). This report presents evidence that acetyl-coa synthetase has two types of requirements for divalent cations. Future studies will determine whether this phenomenon is characteristic of other synthetase reactions having a similar reaction mechanism. EXPERMENTAL PROCEDURE Assays-The activity of acetyl-coa synthetase in the over-all reaction was measured by the rate of disappearance of free coenzyme A in an acetate-dependent reaction. Assay conditions were those of Mahler, Wakil, and Bock (5) as modified and * Supported by Grant AM-06853 from the United States Public Health Service. A preliminary report of this work was published in Federation Proc., 24, 285 (1965). t Research Career Development Awardee 5-K3-HE-13983, United States Public Health Service. described by Webster (3) ; the sulfhydryl group of free CoA was determined by the nitroprusside technique of Grunert and Phillips (6). Protein concentrations were measured by the method of Gornall, Bardawill, and David with crystalline bovine plasma albumin being used as the standard (7). One unit of enzymatic activity catalyzes the disappearance of 1 pmole of CoA per min at 37. Specific activities are expressed in units of enzymatic activity per mg of protein. One unit of acetyl-coa synthetase is equivalent to 0.883 mrmole of enzyme assuming that a homogeneous preparation has a specific activity of 35.4 units per mg of protein and a molecular weight of 32,000 (3). Acetyl-1-14C-adenylate was isolated under equilibrium conditions from partial reaction mixtures containing substrate quantities of enzyme in addition to other appropriate reactants; assay conditions, preparation of carrier acetyladenylate, and the chromatography of acetyl-14c-adenylate (product) on Dowex 1 (Cl-) have been described in detail (4). An aliquot from each reaction mixture was placed on a Dowex 1 (Cl-) column and washed with ice water until no radioactivity was detected in the eluate and then the product was eluted with 25 mm HCl. Thus, contamination of the adenylate fractions by metal chelates of acetate-14c was minimized. Materials and Methods-Acetyl-CoA synthetase was prepared from beef heart mitochondria by the method described by Webster (3). Activities of the various preparations employed are specified in the charts and tables. The chelating resin, Chelex 100 (Na+) was a product of Bio- Rad Laboratories. This resin was converted to the ammonium or potassium form and washed thoroughly with quartz-distilled water prior to use. With the exception of divalent metal salts, reagents used in the assays were stirred with resin for 30 min; potassium chloride was treated with resin in the K+ rather than the NHd+ form. For preparation of metal-poor enzyme, the protein was diluted to a concentration of 2 to 4 mg per ml with 0.02 M KHCO, and stirred with 0.1 volume of Chelex 100 (NH4+) for 30 min at 0. This procedure resulted in a loss of 0 to 20% of the enzymatic activity when nonresin-treated reagents were employed for the assay. The chelating resin was found to contain an agent or agents capable of oxidizing certain divalent metal cations, e.g. Co++, Fe++. Therefore, salts of these metals were dissolved in quartzdistilled instead of resin-treated water. Control experiments with the quartz-distilled water were performed when such oxidizable metal ions were employed. 4164

November 1965 L. T. Webster, Jr. 4165 Double requirement for divalent metal ions in the acetyl-coa synthetase reaction Type is optimally satisfied by Mg++, Mn++, Fe++, Co++, or Ca++ at greater than m&f concent.rations; type is fulfilled by Fe++, Ni++, Cd++, or Cu++ at concentrations only slightly exceeding that of the enzyme. See text for further details. Over-all Partial reaction reactions Acetate + ATP + CoA, types and K acetyl-coa + AMP + PPi CoA types and + Acetate + ATP + E. E(acetyl-AMP) + PPi type 11 acetyl-coa + AMP + E Dialysis tubing (Visking Company) was boiled in three changes of 0.02 ill KHC03-0.5 mm EDTA, washed with water, and dried prior to use. For the experiments of Table V both dialyzed enzyme and an ash of the dialysate from the enzyme were prepared. A second ammonium sulfate precipitate of acetyl-coa synthetase (specific activity, 2.5; 2 to 4 mg of protein per ml) was dialyzed against 0.02 M KHC03-0.08 M KCl-3 mm mercaptoethanol for 1 hour at 0 to make up the dialyzed enzyme. An ash of the dialysate was prepared after dialyzing 10 to 15 mg of the above enzyme preparation in 1 ml against 3 ml of 0.02 M KHC03 for 16 hours at 4. Measured aliquots of the dialysate were heated at 400 for 12 hours in a porcelain crucible and the cooled residue was dissolved in resin-treated water equal to the original volume. Acetyl-1-i4C-CoA and sodium acetate-1-14c were products of New England Nuclear. Prior to use, acetyl-l-r4c-coa was purified by chromatography on DEAE-cellulose (4). Dipotassium adenosine triphosphate and adenylic acid were purchased from P-L Biochemicals. Other chemicals were reagent grade. 0 X 0.4 0 0 2 4 6 [MgC12]- MOLARTY X O3 FG. 1. The effect of increasing magnesium ion concentrations on the acetate-dependent rate of acetyl-coa formation. n a total volume of 0.25 ml, complete reaction mixtures contained the molarities of MgCls shown, 25 pmoles of Tris-HCl buffer, ph 8.0, 0.6 pmole of dipotassium ATP, 37.5 pmoles of KCl, 1 rmole KBH4, 0.35 pmole of free CoA, and 0.75 pmole of potassium acetate. Enzyme added (specific activity, 15 to 30) was adjusted so that the concentration of acetyl-coa formed in the 3-min incubation period at 37 did not exceed 67% of the initial concentration of the rate-limiting divalent cation. Kinetic constants for divalent metal ions in the acetyl-coa synthetase reaction* Assay conditions for the apparent K,,, values are those described under Fig. 1. The rate of acetyl-coa formation at a concentration of 0.8 mm of each divalent metal ion listed was multiplied by 2 to obtain the apparent l,,,. The Mar found for MgClt was arbitrarily assigned a value of 100% for comparison purposes. Cation Mg++ Mn++ Fe++ co++ Ca++ M 7 x 10-d 8 x 10-d 6 X 1O-4 7 x 10-d 9 x 10-J Relative % 100 100 100 85 10 * Kinetic constants for Fe++ and Co++ were determined in the presence of 4 mm KBH, but the figures are not corrected for valence changes of metal ions which may have occurred during the reaction. RESULTS Two types of requirements for divalent cations were found which were arbitrarily classified as type and type. The site of action of each type is summarized in Table and the experimental evidence is presented below. Requirement for Mg++, Mn++, Fe++, Co++, or Ca++ (Type )- Kinetic studies revealed an absolute dependence of the over-all reaction for Mg++ (Fig. 1) or for several cations which could substitute for Mg++ including Mn++, Fe++, Co++, and Ca++. Concentrations of cation exceeding mm were needed for optimal satisfaction of this requirement which was demonstrable without special treatment of the enzyme or reactants. Metal ions which did not stimulate the rate of acetyl-coa formation in this system included 4 mm Ni++, Cd++, Be++, Cr++, Sr++, Zn++, Pb+f, or Fe+++. Apparent K, values for this first type of divalent cation requirement were approximately the same (6 to 9 X 10m4 M) for each of the active cations (Table ). When tested at Km(app) concentrations, Mg++, Mn++, and Fe++ all gave the same calculated V,,, whereas Co++ had about 85 and Ca+f 10% of this value. Appreciable inhibition was noted with 6 mm Mg++; Mn++, or Fe++; Co++ and Gaff were not tested at 6 mm but gave no inhibition at 3 mm concentrations. The influence of 4 mrvr Mg++ on the yield of acetyladenylate under equilibrium conditions is shown in Table. When MgCl* was omitted from reaction mixtures containing highly purified enzyme, no adenylate was formed from acetate and Vm,,

4166 Acetyl-CoA Xynthetase Reaction. Vol. 240, No. 11 V Effect of 4 rnm MgCls on acetyladenylate formation with highly puri$ed acetyl-coa synthetase and either acetate and ATP or acetyl-coa and AMP After all additions, reaction mixture contained 29 pmoles of phosphate (K+) buffer, ph 7.4, 50 pmoles of KCl, 0.25 pmole of ATP (K+), 10 mpmoles of NiC12, 5.0 mpmoles of acetate-l-l% (30,000 cpm per mpmole), and 5.5 units (4.9 mpmoles) of resintreated enzyme, specific activity, 20.7, in a total volume of 0.9 ml. Reaction mixture contained 40 amoles of KHCOa, 0.15 pmole of AMP (K+), 10 mpmoles of NiC12, 5 mv.moles of acetyl-1-w CoA (34,000 cpm per mpmole), and 6.0 units (5.3 mpmoles) of resin-treated enzyme, specific activity, 21.0, in a total volume of 0.9 ml. n each case, the enzyme was preincubated for 2 min at 0 with the radioactive substrate, added to the remaining components except NiC12, and incubated at 37 for i min. Then NiCL was added and the reaction was terminated after another minute of incubation by 0.1 ml of 25% trichloroacetic acid. Acetyl-W-adenylate was isolated as previously described (4). Reaction mixture AcetyPC-adenylate CPm n/.mde. Acetate + ATP + enzyme 330 NO.01 + Mg++ 9,900 0.33 - Ni++ + Mg++ 1,480 0.05 - Enzyme + Mg++ 300-0.01. Acetyl-CoA + AMP + enzyme + Mg++ - AMP - Ni++ - Enzyme 11,200 10,200 360 1,350 340 0.33 0.31-0.01 0.04-0.01 Evidence for heat-stable dialyzable factor required to form acetyladenylate from acetate, ATP, MgCL, and partially purijied acetyl-coa synthetase n a total volume of 0.9 ml, complete reaction mixtures con tained 29 rmoles of phosphate (K+) buffer, ph 7.4, 3.6 @moles of MgC12, 0.25 pmole of ATP (K+), 50 pmoles of KC, 5.0 mpmoles of sodium acetate-l-w (30,000 cpm per mpmole), and 3.3 units (2.9 mpmoles) of a second ammonium sulfate precipitate of acetyl-coa synthetase, specific activity, 2.4 (3). The dialysate was prepared from the enzyme as outlined in the text, and 0.3 ml (or an equivalent volume of an ash of the dialysate) was included in the 0.9-ml incubation mixture where indicated. Mixtures were incubated for 1 min at 37 after which 0.1 ml of 25% trichloroacetic acid was added; acetyl- Gadenylate was isolated as described previously (4). Reaction mixture Undialyzed enzyme. Dialyzed enzyme.. Dialyzed, enzyme + dialysate. Dialyzed enzyme + ashed dialysate... Dialyzed enzyme + resintreated ashed dialysate Dialyzed enzyme + ashed dialysate in EDTA (0.5 mm) Dialyzed enzyme + ashed 0.02 M KHCOZ- (NH4) 804. No enzyme. AcetyPC-adenylate c9m m/bmole 6960 0.23 3120 0.10 6540 0.22 6780 0.23 3690 0.12 3450 0.12 3270 0.11 540 <0.02 ATP. n contrast, addition of MgClz was not required nor did it enhance acetyladenylate yields from acetyl-coa and AMP. Requirement for Fe++, Ni++, Cd++, or Cu++ (Type )-An additional divalent cation requirement for the acetyl-coa synthetase reaction was first suspected because yields of enzymebound acetyladenylate from acetate, ATP, and MgClz were greater with partially purified undialyzed enzyme than with more highly purified but dialyzed preparations. Studies with partially purified but undialyzed enzyme showed that the yields of acetyladenylate under equilibrium conditions in the forward partial reaction could be decreased about 50% by dialysis (Table V). Activity was restored by adding back the dialysate before or after it was ashed. No stimulation of adenylate yields was found with preparations of ashed dialysate which had been treated with EDTA (0.5 mm) or chelating resin. Treatment of highly purified acetyl-coa synthetase with chelating resin markedly decreased the yields of acetyladenylate at equilibrium both from acetate, ATP, and MgC& in the forward partial reaction, and from acetyl-coa and AMP in the reverse partial reaction (Tables V and V). Preincubation of substrate quantities of resin-treated enzyme (approximately 6 mpmoles) with 10 mpmoles of either divalent iron, nickel, cadmium, or copper chloride resulted in markedly increased yields of acetyladenylate both in the forward and reverse partial reactions. Metal ions which were inactive at 10 PM in these systems included Mg++, M&f, Sn++, Cr+f, Co++, Be++, Sr+f, Ca+f, Zn++, and Fe+++. Essentially the same stimulated yields of acetyladenylate were obtained from acetate, ATP, MgC&, and Ni++-treated enzyme when incubation periods were increased from + to 6 min, V Evidence for divalent metal ion requirement in addition to Mg++ for acetyladenylate formation from acetate, ATP, MgC12, and highly puri$ed acetyl-coa synthetase Reaction mixtures, except for enzyme, were the same as described for Table. For metal addition studies highly purified acetyl-coa synthetase (0.1 ml containing 6 units (5.3 m/lmoles) of resin-treated enzyme, specific activity, 33.9) was preincubated for 2 min at 0 with 0.1 ml (10 mpmoles) of a divalent metal chloride where indicated. The 0.2-ml cation-enzyme mixture was then added to the other reaction components to make up the final volume of 0.9 ml. After incubation at 37 for 1 min, reactions were terminated with 0.1 ml of 25% trichloroacetic acid; acetyl- Gadenylate was isolated as described previously (4). Reaction mixture AcetyPC-adenylate Untreated enzyme Resin-treated enzyme + Fe++ + Ni++ + Cd++ + cu++ + Other polyvalent cations* No enzyme + 10 mpmoles of divalent cation7 cpm 14,880 1,130 16,500 16,000 13,400 12,200 920-l ) 400 380-590 mpmole 0.50 0.04 0.55 0.53 0.45 0.41 0.05 * Chlorides of Mgff, Mn++, Sn++, Cr++, Co++, Be++, Sr++, Ca++, or Fe+++. t Chlorides of Fe++, Ni++, Cd++, or Cu++. 0.02

isovember 1965 L. T. Webster, Jr. 4167 indicating that the reaction approached equilibrium. Adding reaction components in a different sequence, i.e. preincubating the enzyme with acetate-w and completing the reaction mixture with NiClz produced increased yields of adenylate, also. ncreasing concentrations of NiClz from 1 X lop5 to 2 X 10e4 M in such a reaction mixture was not accompanied by further increases in acetyladenylat.e, demonstrating that the lower concentration of cation was sufficient to saturate the system. The Fe++, Ni++, Cd++, or Cu++-stimulated formation of acetyladenylate could be shown in the forward partial reaction only when the first type of requirement for divalent metal ions was satisfied, e.g. when reaction mixtures contained 4 mm Mg++ (Table ). n contrast, Mg++ or Mn++ ions were not required for Fe++, Ni+f, Cd++, or Cu++-stimulated formation of acetyladenylate from acetyl-coa and AMP (Tables and V). The type requirement for divalent cations in the partial reactions was confirmed by kinetic studies of acetyl-coa formation in the over-all reaction (Table V). The enzyme and reagents (except MgClz or MnCLJ had to be treated with resin before the rate of acetyl-coa formation was depressed sufficiently to note this effect. Then, in the presence of 3 mm magnesium ions, either Fe++, Ni++, Cd++, or Cu++ (3 to 5 X 1OW M) increased the rate of acetyl-coa formation. All four of these divalent cations were still stimulatory when 3 mm MnC12 was substituted for magnesium ions to satisfy the type requirement for divalent metal ions. Cations which did not stimulate acetyl- CoA formation in this system included Mg++, Mn++, Cr*, Co++, Be++, Sr++, Ca++, Zn++, and Fe+++ at a concentration of 7 PM. Evidence for divalent metal ion requirement for acetyladenylate formation from acetyl coenzyme A, AMP, and highly pur$ed acetyl-coa synthetase n a total volume of 0.9 ml, complete reaction mixtures contained 40 pmoles of potassium bicarbonate, 0.15 pmole of AMP (K+), 5 mpmoles of acetyl-1- GCoA (34,000 cpm per mpmole), and enzyme. For metal studies, highly purified acetyl-coa synthetase (0.1 ml containing 6.4 units (5.7 mpmoles) of resintreated enzyme; specific activity, 30.9) was preincubated for 2 min at 0 with 0.1 ml (10 mrmoles) of the divalent metal chloride. The 0.2-ml cation-enzyme mixture was then added to the other reaction components to make up the final volume of 0.9 ml. After incubation at 37 for 1 min, reactions were terminated with 0.1 ml of 25% trichloroacetic acid; acetyl-14c-adenylate was isolated as previously described (4). Reaction mixture Untreated enzyme Resin-treated enzyme + Fe++ + Ni++ + Cd++ + cu++ + Other polyvalent cations* V Acetyl-W-adenylate cm??%p?%& 12,100 0.36 1,320 0.04 11,200 0.33 11,800 0.35 9,200 0.27 8,300 0.24 l,loo-1,400-0.04 No enzyme + 10 mbmoles of diva- 340-420 -0.01 lent cation! - * Chlorides of Mg++, Mn++, Sri++, Cr++, CO++, Be++, Sr++, Ca++, or Fe+++. t Chlorides of Fe++, Ni++, Cd++, or Cu++. E$ect of certain divalent metal ions on acetate-dependent rate of acetyl-coa formation in presence of 3 mu MgClz or MnC12 Complete reaction mixtures contained 25 pmoles of Tris-HC buffer, ph 8.0, 0.75 pmole of MgClz or MnCL as indicated, 0.6 pmole of dipotassium ATP, 37.5 pmoles of KC, 0.35 rmole of CoA, 0.75 rmole of potassium acetate, 1 pmole of KBH, and 0.004 to 0.7 mg of enzyme in a total volume of 0.25 ml. Complete and control (- acetate) mixtures were incubated for 3 min at 37 after which reactions were terminated with 0.06 ml of 3Oyc metaphosphoric acid and CoA disappearance was determined (3). The effect of added cations was tested by preincubating the resin-treated enzyme (protein concentration, 2 to 4 mg per ml) at 0 for 2 min in 5 X low5 M of the metal salt indicated. The mixture was then diluted with resin-treated 0.02 M KHC03 to achieve a protein concentration appropriate for assay. Final concentrations of preincubated divalent cation in the incubation mixtures ranged from as high as 7 PM for the inactive species to as low as 0.3 PM for some of the active divalent metal ions (Fe++, Ni++, Cd++, or Cu++). system Untreated enzyme and reagents. Untreated enzyme and resin-treated reagents*... Resin-treated enzyme and reagents*. Resin-treated reagents*: resin-treated enzyme preincubated with FeCls. NiC12. CdClz. CuClz. Other cations?. Resin-treated reagents :* resin-treated enzyme preincubated with chlorides of Fe++, Ni++, Cd++, or Cu++; magnesium and manganese chloride omitted from reaction mixture. V -- Acetyl-CoA MgClz formed MnCh 17.1 17.7 15.6 15.9 1.4-1.6 1.4-1.6 14.4 10.5 11.9 8.7 13.2 13.5 8.0 7.2 1.2-2.1 1.2-2.1 < 1 * All reagents were treated with resin except added MgClz or MnCll. t Chlorides of Mg++, Mn++, Cr++, Co++, Be++, Sr++, Ca++, Zn++, or Fe+++. nhibition by ZnCln and CaC&Zinc ions at 0.3 mm inhibited the over-all reaction about 75% in the presence of 4 mm Mg++. ncreasing the concentration of Mg+f to 12 mm or adding the chloride salts of Ni++ or Cd++ to concentrations as high as 6 m&r did not affect this inhibition. Calcium ions at concentrations of 6 mm inhibited the over-all reaction about 50% in the presence of 4 mm Mg++ or Mn++. Doubling the Mg++ or Mn+f concentrations reversed the inhibition produced by Ca++ whereas addition of Ni++ or Cd++ chlorides (4 mm final concentration) did not. DSCUSSON The results described demonstrate two types of requirements for divalent cations in the acetyl-coa synthetase reaction (Table ). n the first type, the metal ion (Mg++, Mn++, Co++, Fe++, or Ca++) is required in relatively high concentrations ( > mm) for maximal enzymatic activity in the over-all reaction

Acetyl-CoA Xynthetase Reaction. Vol. 240, No. 11 (Fig. 1, Table ) and also for formation of enzyme-bound acetyladenylate from enzyme, acetate, and ATP (Table ). These cations probably are not needed to bind acetyl-coa, AMP, or acetyladenylate to the enzyme because their addition is not essential for the formation of acetyladenylate from enzyme, acetyl-coa, and AMP (Tables and V). The possibility that a cation in this group is bound to highly purified and resintreated acetyl-coa synthetase in sufficient quantities to promote the reverse partial reaction appears unlikely because the same enzymatic preparation displayed an absolute dependence on added magnesium ions for formation of acetyladenylate from acetate and ATP in the forward partial reaction (Table ). f they are not essential for binding of acetyladenylate to the enzyme, the most plausible explanation is that cations in the first category are necessary to chelate ATP and pyrophosphate. All of the KmcaBP) values for metal ions in the over-all reaction are near mm which is close to that of 0.9 mm found previously for ATP (3). n magnetic resonance studies of metal activation of certain kinase reactions involving cleavage of ATP into ADP and inorganic phosphate, Cohn s group has presented evidence that chelation of ATP is involved where there is a broad divalent cation specificity and calcium ions, in particular, are active (1). Furthermore, Berg has demonstrated that Mg++ is required for formation of ATP from chemically synthesized acetyladenylate, pyrophosphate, and an acetyl-coa synthetase from yeast (8). The second type of requirement for divalent metal ions has several features which distinguish it from the first. With the exception of iron, the spectrum of active ions is different (Ni++, Cd++ and CL?). Divalent ions in the second category appear to be Closely associated with the enzyme which had to be dialyzed or treated with a chelating resin before the metal requirement was demonstrated (Table V). Removal of some of the metal ions by such a mild procedure as dialysis suggests that their bonding to the enzyme is not extremely strong. However, appreciable affinity must exist between the cation and enzyme because enhanced yields of acetyladenylate were obtained with final concentrations of metal ions approximately 2-fold those of the enzyme (Tables V and V). The last experiments do not provide information concerning the stoichiometry between metal ions and enzyme because only about 5 to 11% of the isotopic substrate and enzyme were present under equilibrium conditions as acetyladenylate enzyme complex. Divalent metal ions in the second category were essential for formation of acetyladenylate in both partial reactions, suggesting that they were involved in binding of certain substrate or substrates to the enzyme (Tables V and V). At neutral ph, the substrates and acetyladenylate are negatively charged; therefore, a divalent cation could be involved in attracting any or all of these compounds to the enzyme. Regardless of which substrates are found to be ligands of the divalent metal, a different type of mechanism is involved for the acetyl-coa synthetase reaction than shown previously for the ADPeP; kinases. When a divalent cation is required for binding substrate to enzyme in the latter cases, the divalent metal ion specificity is quite narrow and calcium is inhibitory (1). n contrast, several cations presumably are capable of binding to acetyl- CoA synthetase, and although high concentrations of Ca++ inhibit the over-all reaction, this inhibition is reversed by Mg+f or Mn++ rather than by the metal ions which bind to the enzyme. Acetyl-CoA synthetase from bovine heart mitochondria also displays the type requirement for divalent cations. -4t present there is no evidence as to which divalent metal ion is attached to acetyl-coa synthetase in its native state nor as to what the stoichiometry is between metal and enzyme. Knowledge of the approximate molecular weight of acetyl-coa synthetase has been obtained recently (3) and this, in combination with studies of metal binding to enzyme in the presence and absence of substrates and inhibitors, may provide answers as to which and how much cation is naturally bound and whether the bound metal ions are located at the active site of the enzyme. The double requirement for divalent ions in the acetyl-coa synthetase reaction may reconcile the proposal of ngraham and Green with that of Berg. The former investigators advocated a role for metal in binding of an intermediate to the enzyme whereas Berg felt that magnesium ions were concerned primarily with the chelation of ATP (S-11). The double requirement may also be found for some of the other synthetase reactions in which ATP is cleaved to AMP and pyrophosphate with the intermediate formation of an enzyme-bound acyladenylate. Examples of enzymes from which acyladenylates have been isolated include firefly luciferase, and several aminoacyl-rna and fatty acyl-coa synthetases (4, 12-19). SUMMARY A double divalent cation requirement was found for the acetyl coenzyme A synthetase reaction in which acetate, adenosine triphosphate, and CoA react to form acetyl-coa. Adenosine triphosphate is cleaved to pyrophosphate and adenosine monophosphate with enzyme-bound acetyladenylate being formed as an intermediate. Concentrations greater than mm of either magnesium, manganese, iron, cobalt, or calcium ions are needed for maximal rates of acetyl-coa formation in the over-all reaction. Magnesium ions stimulate the first partial reaction, i.e. formation of enzyme-bound acetyladenylate from acetate and ATP, but Mg++ is not required for the reverse partial reaction, i.e. the formation of acetyladenylate from acetyl-coa and AMP. This type of divalent cation dependence can be shown without special treatment of enzyme and reactants. The second type of divalent metal requirement is easily demonstrated only after acetyl-coa synthetase is dialyzed or treated with a chelating resin. Thus, in addition to optimal concentrations of divalent metal ions of the first type, e.g. Mg+f, the overall reaction requires either divalent iron, nickel, cadmium, or copper ions in concentrations only slightly exceeding those of the enzyme. One of these four divalent metal ions is necessary for formation of enzyme-bound acetyladenylate from acetyl-coa and AMP and from acetate and ATP (the partial reaction involving acetate and ATP also requires a divalent cation of the first type such as Mg++). The results are consistent with cations in the first group (Mg++) being needed to chelate ATP whereas the metal ions in the second category are essential for binding substrate or substrates to the enzyme. Acknowledgment-The author wishes to thank Mr. Douglas Morrison for his technical assistance. REFERENCES 1. COHN, M., Biochemistry, 2, 623 (1963). 2. BOYER, P. D., LARDY, H., AND MYRB~CK, K. (Editors), The enzymes, Vol. V, Academic Press, nc., New York, 1962. 3. WEBSTER, L. T., JR., J. Biol. Chem., 240, 4158 (1965). 4. WEBSTER, L. T., JR., J. Biol. Chem., 238, 4010 (1963).

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