Transient Kinetic Studies of Fatty Acid Synthetase A KINETIC SELF-EDITING MECHANISM FOR THE LOADING OF ACETYL AND MALONYL RESIDUES AND THE ROLE OF COENZYME A*

A kinetic self-editing mechanism for correcting er- rors in the loading of thioester substrates is described for the animal fatty acid synthetase reaction. In the catalyzed reaction, these substrates load competitively on a common phosphopantetheine site, and during each of the eight loading steps the enzyme sites are parti- tioned between competent and incompetent substrate molecules. The incompetently bound substrate is re- moved by CoA through reversal of the loading reaction and partitioning again occurs. The loading-unloading cycle is repeated until competent enzyme complex is formed and the reaction proceeds. Furthermore, at each step the loading of a malonyl residue is competitively favored as is the unloading of enzyme-bound acetyl groups. This mechanism is entirely consistent with the re- cently postulated role (Stern, A., Sedgwick, B., and Smith, S. J. Biol. a co-substrate. Supporting evidence is obtained by monitoring the progress curves of NADPH oxidation by chicken liver fatty acid synthetase in the stopped flow apparatus. At noninhibiting acetyl-coA, the re- action shows an initial lag period as the result of preferential formation of malonyl-enzyme and time-de- pendent recycling of the loading step to obtain competent acetyl-enzyme. At a malonyl-CoA/acetyl-CoA ratio of 2:1, the induction time of Steady state parameters of reactions catalyzed by fatty acid synthetase The reactions were carried out in a conventional Gilford spectrophotometer at 37 “C, pH 7.0, as described under “Experimental Procedures.” FAS, fatty acid synthetase.

A kinetic self-editing mechanism for correcting errors in the loading of thioester substrates is described for the animal fatty acid synthetase reaction. In the catalyzed reaction, these substrates load competitively on a common phosphopantetheine site, and during each of the eight loading steps the enzyme sites are partitioned between competent and incompetent substrate molecules. The incompetently bound substrate is removed by CoA through reversal of the loading reaction and partitioning again occurs. The loading-unloading cycle is repeated until competent enzyme complex is formed and the reaction proceeds. Furthermore, at each step the loading of a malonyl residue is competitively favored as is the unloading of enzyme-bound acetyl groups.
This mechanism is entirely consistent with the recently postulated role (Stern, A., Sedgwick, B., and Smith, S. J. Biol. Chem. (1982) 257,799-803) of CoA as a co-substrate. Supporting evidence is obtained by monitoring the progress curves of NADPH oxidation by chicken liver fatty acid synthetase in the stopped flow apparatus. At noninhibiting acetyl-coA, the reaction shows an initial lag period as the result of preferential formation of malonyl-enzyme and time-dependent recycling of the loading step to obtain competent acetyl-enzyme. At a malonyl-CoA/acetyl-CoA ratio of 2:1, the induction time of the reaction is 1.02 2 0.05 s at 6 "C. It decreases with increasing acetyl-coA concentration or preincubation of the enzyme with acetyl-coA which promotes acetyl-enzyme formation but is slightly increased upon preincubation with malonyl-CoA. Increasing acetyl-coA causes a parallel decrease in steady state cycle time (i.e. the average time required to complete a single malonyl-CoA condensation cycle), suggesting that the latter is limited by the lag period. At inhibitory acetyl-coA, the steady state cycle time is lengthened due to acetyl-enzyme formation at malonyl-CoA loading steps and to the recycling necessary to obtain competent malonyl-enzyme.
A requirement of CoA for the first condensation cycle is unequivocally demonstrated in conventional spectrophometric assays and stopped flow experiments by * This work was supported by National Institutes of Health Grant AM 13390. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. )I To whom correspondence should be addressed.
using phosphotransacetylase and acetyl phosphate as a CoA trap. This requirement at each loading step is normally met by CoA generated through initial loading. At noninhibitory acetyl-coA, added CoA inhibits the reaction and slightly increases the lag. At inhibitory acetyl-coA, a low concentration of CoA increases the lag period by removing competently bound acetyl residues, while the steady state cycle time is decreased owing to the preferential removal of incompetently bound acetyl residues at each of the malonyl-CoA loading steps. At very high CoA, the lag period and steady state cycle time are increased by indiscriminate unloading and by competitive inhibition of noncovalent binding of both substrates.
In recent reports, the fatty acid synthetase reaction has been shown to require CoA as a co-substrate in catalytic amounts (1-5). The role of CoA was thought to be as the acyl acceptor in the termination step of the reaction (1, 2) or alternatively in substrate loading steps to remove inappropriately bound acetylor malonyl-residues in abortive complexes formed through competitive loading of these substrates (3, 4). In the present study, stopped flow experiments are undertaken to determine the transient behavior of this reaction and the effect of CoA. Progress curves of NADPH oxidation obtained at noninhibitory acetyl-coA show an initial lag period due to preferential formation of malonyl-enzyme and time-dependent repetition of unloading-loading to acquire competent acetyl-enzyme. The lag period and steady state cycle time calculated from the steady state rate are affected by changing the acetyl-CoA/malonyl-CoA ratio and by preincubation of synthetase with either thioester substrate in a manner entirely consistent with the latter interpretation. A kinetic self-editing mechanism for correcting errors in the loading of substrates is delineated. A preliminary report of this work has been published (6). commercial malonyl-CoA was purified chromatographically as described by Hsu et al. (8). As expected from the early data of Wakil and Ganguily (9), acetyl-coA was required for fatty acid synthesis with purified malonyl-CoA.
Purification of Chicken Liver Fatty Acid Synthetase Fatty acid synthetase was purified from male Hubbard grandparent chickens, kindly provided by A w a y Farms a t 2 days of age, which were killed a t 4-6 weeks of age following fasting and refeeding. The enzyme was isolated by modification of the original procedure of Hsu and Yun (10). All purification steps were carried out a t 4 "C instead of room temperature to minimize proteolysis, and the buffers contained 1 mM DTT and 1 mM EDTA unless otherwise specified. The ammonium sulfate fractionation and calcium phosphate gel adsorption steps were performed as before, except the dialysis step prior to gel treatment was eliminated. During DEAE-cellulose chromatography, the column loaded with fatty acid synthetase was successiuely eluted with 0.04, 0.075, and 0.14 M potassium phosphate buffer, pH 7.0. A minor activity peak (peak I) was obtained in the 0.075 M buffer, whereas the bulk of enzyme (peak 11) was eluted by 0.14 M buffer. Each peak was collected and concentrated by precipitation with 40% saturated ammonium sulfate. The pellet was dissolved in 40 ml of 0.05 M potassium phosphate buffer pH 7.0, containing 1 mM DTT and 3 mM EDTA, loaded on a blue Sepharose column (3 X 10 cm) equilibrated with the same buffer, and washed with buffer to remove contaminating proteins. Fatty acid synthetase was eluted with this buffer containing 0.3 M NaC1. The purified enzyme was concentrated with ammonium sulfate as before and stored in 0.2 M potassium phosphate buffer, pH 7.0, containing 10 mM DTT, 3 mM EDTA, and 10% (v/v) glycerol a t -70 "C. Under these conditions, the enzyme was completely stable for 1 year. The presence of two enzymatically active peaks confirmed earlier data from this laboratory (10). Peak I1 had a yield of 45% and a specific activity of 160 f 20 nmol of acetyl-CoA incorporated per min/mgof protein a t 37 "C. It was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis ( Fig. 1) and is routinely used in the present study.
Before each experiment, the enzyme was equilibrated with the appropriate buffer by passage through a Sephadex PD-10 column. The specific activity and kinetic parameters as well as fatty acid product pattern of peak I were comparable to those of peak I1 (i.e. 67% palmitic and 11% stearic acids). Furthermore, rechromatography of peak I on a DEAEcellulose column yielded a bimodal elution pattern similar to that obtained for the unresolved enzyme, indicating interconvertability between two forms. The reason for this behavior is not clear. However, the possibility of conformational isomers deserves consideration.
The concentration of purified fatty acid synthetase was determined at 279 nm using a dimer molecular weight of 500,000 (1 1).

Steady State Kinetic Assays of Fatty Acid Synthetase Actioities
The kinetic parameters of fatty acid synthetase activities were obtained by initial velocity measurements of NADPH oxidation a t 340 nm in a Gilford 250 or a Cary 16 spectrophotometer. The reaction mixtures contained 0.15 M potassium phosphate buffer, pH 7.0, 1 mM DTT, 3 mM EDTA, and appropriate substrates and enzyme as indicated below in a volume of 1.0 ml. The temperature was thermostatically regulated a t 37 "C unless otherwise specified. In all cases, velocities were corrected for endogenous NADPH oxidation of a control without enzyme. Substrate and enzyme concentrations for the acetyl-CoA-primed reaction, and the ketoreductase reaction with acetoacetyl-CoA or S-acetoacetyl-N-acetylcysteamine were , respectively, acetyl-coA, 0.22-14.4 pM, malonyl-CoA, 0.43-11.5 pM, NADPH, 140 p~, chicken liver fatty acid synthetase, 2-3 pg; acetoacetyl-coA, 3.8-91 p~, NADPH, 75 p M , chicken liver fatty acid synthetase, 100 pg; S-acetoacetyl-N-acetylcysteamine, 0.9-10 mM, NADPH, 75 p~, chicken liver fatty acid synthetase, 4-5 pg. Kinetic parameters of the acetyl-CoA-primed reaction were determined by fitting initial velocity data to an equation for a ping-pong mechanism with double competitive substrate inhibition Lineweaver-Burk analysis. The concentrations of substrates used in steady state and stopped flow experiments were determined by absorption measurements according to the following molar extinction coefficients: acetyl-coA and acetoacetyl-CoA, t = 15,400 M-' cm" at 259 nm, pH 7.0; malonyl-CoA and CoA, t = 14,600 cm" at 260 nm, pH 2.0; S-acetoacetyl-N-acetylcysteamine, t = 4,700 M-l cm-l at pH 7.0 (7); and NADPH, t = 6,200 M-' cm-l at 340 nm, pH 9.0.
In experiments employing a CoA trap, phosphotransacetylase and 5 mM AcPO, were included in each assay. Since inorganic phosphate strongly inhibits the phosphotransacetylase reaction (12), potassium phosphate was replaced by a buffer containing 0.12 M HEPES, pH 7.0, and 20 mM activator KCI. Control experiments indicated that the enzyme was fully active in the presence of either phosphotransacetylase or AcPO, alone. Experimental-The reaction mixtures contained 0.15 M potassium phosphate buffer, p H 7.0, 1 mM DTT, 3 mM EDTA, and appropriate amounts of substrates and fatty acid synthetase as indicated in legends. In experiments using the CoA trap, phosphotransacetylase and AcP04 were present and phosphate buffer was replaced by 0.12 M HEPES and 20 mM KC1 as described under "Steady State Kinetic Assays of Fatty Acid Synthetase Activities." Reagent solutions were prepared fresh daily, kept on ice (or at room temperature for solutions containing enzyme), and degassed immediately before use. The enzyme solution was stable during the course of each experimental period of up to 4 h. The reactions were monitored by following NADPH oxidation a t 340 nm and a thermostatically controlled temperature of 6 "C except as otherwise noted.
In order to minimize artifacts due to time-dependtnt diffusion of reagents, each recorded experiment was preceded by a nonrecorded prerun. The zero time absorption was corrected by appropriate reagent blanks obtained prior to each experiment. Endogenous oxidation of NADPH in the blank without enzyme was not detectable in these short term experiments. The progress curves of stopped flow experiments were highly reproducible. The results shown are averages of a minimum of five runs/experiment repeated a t least once with different enzyme preparations.

RESULTS
Initial Rate Profiles of Fatty Acid Synthesis from Acetyland Malonyl-CoA-In these experiments, the fatty acid synthesis reaction was monitored in the stopped flow apparatus at 340 nm by mixing equal volumes of a solution containing enzyme and NADPH with one containing acetyl-and malonyl-CoA. At noninhibiting concentrations of CoA thioesters in the final reaction mixture, a lag period was observed before NADPH oxidation reached a steady state as shown in a typical experiment (Fig. 2, curve I ) . A value of 1.02 f 0.05 s for T, the induction time (cf . Table I for definitions), was obtained which equals, within experimental error, 50% of the steady state cycle time ( c ) of 1.94 f 0.02 s calculated from the steady state rate (Table I, Experiment 3). This lag of NADPH oxidation is presumably due to the presence of slow process(es) in the first malonyl-CoA condensation cycle. 7 decreases with increasing temperature to 100 ms at 27 "C and 60 ms a t 37 "C, but is relatively unaffected by enzyme concentration between 35 nM and 0.7 p~. In order to maximize the lag period for precise quantitation, subsequent experiments were performed a t 6 "C.
If an experiment is carried out with enzyme and thioester substrates placed in separate syringes as done in Fig. 2, curve 1, a t final concentrations of 3.5 p~ chicken liver fatty acid synthetase, 70 p~ NADPH, 14 pM acetyl-coA, and 28 p M malonyl-CoA, the progress curves (not shown) are similarly shaped. However, a burst of NADPH oxidation is not observed despite the high enzyme concentration employed, suggesting that the slow process occurs prior to the release of the oxidized nucleotide.
In other experiments employing constant malonyl-CoA at 13.5 p~, T is found to decrease with increasing acetyl-coA (Table I, Table I, Experiments 5 and 6). The initial rate profile at 100 p~ acetyl-coA is complex (Fig. 2, curve 2 ) with a relatively slow burst. The burst size cannot be determined but is significantly smaller than the optical density change Progress curves of NADPH oxidation with acetyland malonyl-CoA as co-substrates. Curve I , syringes in the stopped flow apparatus contained SI, chicken liver fatty acid synthetase and NADPH; S1, acetyl-and malonyl-CoA. Final concentrations in the reaction cuvette were, respectively, 0.7, 60, 6.5, and 13.5 p~. T = 1.01 s; c = 1.94 s. Curue 2, conditions were identical with those in curve 1 except that acetyl-coA was 100 p M and c = 5.865 s. curue 3, final concentrations were as those in curue 2 except acetyl-coA was included in Sl to yield preformed acetyl-chicken liver fatty acid synthetase. Contents of SI were equilibrated at 6 "C for less than 10 min prior to use. Hydrolysis of acetate occurred at a very slow rate and had no significant effect on results obtained in the experiment. c = 2.04 s. Curve 4, final concentrations were as those in curve 1 except malonyl-CoA was included in S1 and NADPH in S,. In this experiment, solutions of enzyme and malonyl-CoA were degassed and equilibrated a t 6 "C separately and mixed immediately before use to minimize decarboxylation. Progress curves of the first (' 2 min after mixing) and last (<9 min after mixing) runs were identical within experimental error. 7 = 1.1 s; c = 1.88 s. The spur between 0 and 300 ms is an instrument artifact which does not occur in the same experiment performed on other days. The induction time T (cf. Table  I  (0.0175) calculated for oxidation of two NADPH/enzyme dimer. The presence of this burst indicates that the slow process responsible for the observed lag is not the result of slow addition of substrates or subsequent rate-limiting step(s) in the reaction sequence, and that this process is limiting the rate of the first steady state cycle. The latter conclusion is supported by proportional decreases of T and c (as indicated by a constant T / C ratio of 0.47-0.53; (Table I, Experiments 1, 2, 3, and 5) with a 9-fold increase of noninhibitory acetyl-CoA.
When the experiment in Fig. 2, curve 1 , is repeated by preincubation of acetyl-coA with fatty acid synthetase (Fig.  2, curue 3), a partial burst is again observed and is more prominent than that seen in Figure 2, curve 2, at high acetyl-CoA. A residual lag is also observed, but the steady state cycle time ( Table I versus 7), and a lag period of 0.93 f 0.01 s is regenerated with a decreased steady state cycle time. Preincubation with malonyl-CoA (Fig. 2, curve 4 ) slightly lengthens the lag period of 1.02 f 0.05 s to 1.13 f 0.05 s without an accompanying change in steady state cycle time (Table I, Experiment 10 verus 3). These effects of preincubation on initial rate of NADPH oxidation (i.e. acetyl-coA shortens, and malonyl-CoA lengths the lag) but not on steady-state cycle time confirm the occurrence of a slow process as an early event in the reaction. Furthermore, this process may be identified with the time-dependent reordering of substrate binding for competency in catalysis.
In the course of this study, it was found that commercial malonyl-CoA is contaminated by acetyl-coA despite a claim to the contrary. Smaller amounts of CoA are also present. The acetyl-coA content as analyzed by incubating the commercial product with phosphotransacetylase (10 unitsiml in 0.15 M K phosphate, 10 mM EDTA at pH 7.0) followed by determination of CoA release with 0.5 mM 5,5'-dithiobis(nitrobenzoic acid) is 7 mol %. Key experiments (Table   I, Experiments 3 and 10) were therefore repeated with acetyl-CoA free malonyl-CoA obtained by paper chromatography (cf. see "Experimental Procedures" for details). The lag was  Steady state cycle time, the average time required to complete a single malonyl-CoA condensation cycle. This value is calculated from the steady state rate (i.e. the linear portion of progress curve) from the stopped flow instrument according to c = 2/(NADPH oxidized 5" chicken liver fatty acid synthetase subunit"). For the purpose of this study, the steady state cycle time is used to facilitate comparison with the induction time. It should be noted that the steady state rates determined by stopped flow experiments agree with those obtained in conventional spectrophotometry at the same temperature within experimental error.
Experiments were carried out as in Fig. 2, curue I , except acetyl-CoA and malonyl-CoA concentrations were varied as indicated.
* Experiments were carried out as in Fig. 2, curue 4 . ' Same as in Footnote i except purified malonyl-CoA was used.
mental Procedures") was used.
Experiments were carried out as in Fig. 2, curue I , except CoA Experiments were carried out as in Fig. 6, curve 2.
' Experiments were carried out as in Fig. 6, curue 3. was included in syringe 2.  still present, and the parameters (Table I, Experiments 4 and 11) were identical with those employing impure malonyl-CoA within experimental error.
Acetoacetyl-CoA is reduced by NADPH via ketoreductase partial activity of the enzyme without de novo fatty acid synthesis. The steady state parameters of ketoreductase using this substrate or the alternate substrate S-acetoacetyl-Nacetylcysteamine and the acetyl-CoA-primed reaction as determined by conventional spectrophotometry are shown in Table 11. The maximal rate for the S-acetoacetyl-N-acetylcysteamine reaction is -2 times that for the acetyl-coAprimed reaction, suggesting that the hydride transfer step is not rate-limiting in the latter reaction and is unrelated to the slow process, as expected. As shown in Fig. 3, progress curves of stopped flow experiments employing thioesters of acetoacetate as the only substrate are linear, with no detectable lag.
Effects of CoA on Fatty Acid Synthesis from Acetyl-and Malonyl-CoA-In recent reports, the fatty acid synthetase reaction has been shown to require CoA as a substrate in catalytic amounts (1)(2)(3)(4)(5). Since the observed lag results from slow reordering of enzyme-bound substrate and since the unloading reaction involves CoA, the effects of this coenzyme on the steady state and transient kinetic behavior of the enzyme are examined in the following experiments.
The CoA requirement for chicken liver fatty acid synthetase was established by inclusion of a CoA-trapping system containing phosphotransacetylase and AcPO, in the spectrophotometric assay for overall synthetase activity. NADPH oxidation is inhibited 90 k 5% by this treatment (Fig. 4A). This inhibition is relieved 50% (to 45% of original activity) by addition of 100 p~ potassium phosphate which inhibits the phosphotransacetylase reaction (12). It is also relieved transiently by addition of 19 nM CoA (Fig. 4B). Corresponding stopped flow experiments are shown in Fig. 5. The control curve containing no phosphotransacetylase (Fig. 5A, curve I ) is comparable to Fig. 2, curve 1. In the presence of phosphotransacetylase, (Fig. 5A, curve 2), the lag is infinitely lengthened by the CoA trap, as would be expected if CoA participates in the first malonyl-CoA condensation cycle prior to hydride transfer. Addition of 10 p~ CoA (Fig. 5B, curve 2 versus curve

I ) again relieves inhibition until the coenzyme is depleted by the trap.
Since CoA is a product of the reaction, it is expected to be also inhibitory. The steady state inhibition of fatty acid synthetase by CoA was shown in an early report by Katiyar et al. (14), and more recently by us2 at noninhibitory acetyl-CoA. At 100 p~ acetyl-coA and 13.5 p~ malonyl-CoA where the reaction is strongly inhibited by the former, however, 10 FM CoA relieves and 100 FM CoA promotes this inhibition. Similar results are obtained by Cox and Hammes (15). These effects of CoA are also demonstrated in the stopped flow apparatus. In an experiment carried out as in Fig. 2, curve I , at low acetyl-coA except with 7.5 p~ CoA present (Table I,  Experiment 12 versus 3), the lag is slightly increased with a 27% increase in steady state cycle time. Experiments using 100 p~ acetyl-coA are shown in Fig. 6. Fig. 6, curve I , is a control progress curve without CoA where a burst is evident.
Addition of 10 ~L M CoA (Fig. 6, curue 2 ) regenerates a lag period of T = 0.88 f 0.05 s and decreases the steady state cycle time from 5.58 +. 0.40 to 3.72 k 0.10 s ( Table I, Experiment 13 versus 7), indicating while CoA at this concentration activates the reaction, it lengthens the slow process. Addition of 100 p~ CoA (Fig. 6, curve 3) is inhibitory and further increases T to 2.65 -+ 0.18 s with a concomitant increase in steady state cycle time (6.76 +. 0.18 s; Table I, Experiment 14). DISCUSSION A simplified scheme for the reaction catalyzed by fatty acid synthetase is shown in Fig. 7. The reaction is primed by acetyl-coA which condenses seven times with malonyl-CoA to yield palmitic acid as the final product. For the purpose of clarity, repetitive condensation cycles which differ only in the size of growing acyl chain are represented by a single, generalized cycle (enclosed area a t right) and the hydroxyl acylation site is not shown. Following completion of chain elongation, palmitic acid is released from palmityl-enzyme by deacylation.
The catalytic reaction has an obligatorily ordered kinetic sequence for the covalent addition of substrates. During the first malonyl-CoA condensation cycle, acetylation occurs first (Step l), which is followed by loading of a malonyl residue (Step 3), condensation (Step 4), and reduction of enzymebound acetoacetate to butyrate (Steps 5-7). In subsequent condensation cycles, only malonyl-CoA is required. Early studies (8,14) established that the free, unliganded enzyme binds either substrate in a random manner. In the synthetase reaction when acetyl-coA is added last to initiate NADPH oxidation, a time-dependent lag of initial rate was observed by Lynen (16) who suggested that this is due to malonylenzyme formation and the displacement of enzyme-bound malonyl-residues by acetyl residues which must occur before reaction can proceed. However, participation of CoA was not anticipated.
Recent discovery of a CoA requirement for the synthetase (1) led to the postulation by Sedgwick and Smith (3) Stern et al. (4) of a role of this coenzyme for the removal of inappropriately bound acetyl or malonyl residues to allow access of the enzyme site to the competent substrate. This hypothesis is supported by data obtained in equilibrium binding studies which show rapid and competitive loading of both substrates and that CoA depletion enhances and CoA addition inhibits the extent of loading site occupancy. Critical evidence for such a role in the catalyzed reaction is provided by the transient kinetic experiments performed in this work.
On the basis of current knowledge, a kinetic self-editing mechanism for each of the eight loading reactions (Steps 1, 3, 9, etc.) in Fig. 7 is delineated as shown below.
In http://www.jbc.org/ occurs, and the loading-unloading sequence is repeated until a competent complex is obtained. While loading (Reaction 1 in forward direction) is not limiting the catalytic rate, repetitive partitioning is and is seen as a lag period in the two loading steps (Steps 1 and 3) during the first malonyl-CoA condensation cycle. Moreover, the competent complex derived from either initial or repeated loading is subjected to partitioning in the steady state between unloading by CoA and the forward reaction as determined by the relative magnitude of the rate constants and the CoA concentration. Since CoA activates unloading of both substrates, equilibration between competent and the abortive complexes is accelerated. Increasing CoA has the effect of simultaneously facilitating competent loading by removing the abortive substrate and reducing it by removing the competent substrate, with the net result of either stimulating or hindering a given loading step depending on the relative sensitivity of the bound substrate toward CoA. Since acetyl-coA is required for Step 1 and malonyl-CoA is required for subsequent loading, a stimulating (or inhibitory) effect on the former would be accompanied by an opposite effect on the latter. In addition, since noncovalent association of either acetyl-or malonyl-CoA (step 1; Reaction 1 in forward direction) for the enzyme owes, a t least in part, to the affinity of the CoA group, free CoA would be a potent competitive inhibitor of this step.
In the following discussion, our experimental observations are analyzed on the basis of the kinetic self-editing mechanism. During the loading process, covalent incorporation of malonyl residues is competitively favored. When acetyl-coA is in limited access as in Fig. 2, curve 1 , malonyl-enzyme is formed predominantly and the lag period in NADPH oxidation is primarily due to slow re-equilibration to obtain competent acetyl-enzyme through Step 1. Reduction of the lag period under conditions ( i e . increasing acetyl-coA concentration or preincubation with acetyl-coA; Fig. 2, curves 2 and 3, and Table I) predisposed to acetyl-enzyme formation, but not under conditions ( i e . preincubation with malonyl-CoA) inducing malonyl-enzyme formation, is in agreement with malonyl-CoA being the preferred substrate. Since synthesis of palmitic acid requires seven additions of malonyl-CoA/each acetyl-coA, this property permits the attainment of greater catalytic efficiency. The complex burst shown in experiments with acetyl-coA more readily accessible (Fig. 2, curues 2 and  3) represents combined initial rate profiles of three separate enzyme species. Those molecules contain competently bound acetyl and malonyl residues which yield the burst, and those molecules contain incompetent malonyl (for Step 1) or acetyl (for Step 3) residues which require equilibration resulting in a lag period.
In addition to reducing the lag period, high acetyl-coA induces acetyl-enzyme formation which prevents proper loading of malonyl residues (Steps 3, 9, etc.) and prolongs the steady state cycle time. The reversal of both effects by high malonyl-CoA (Table 1,Experiment 9) further supports the above analysis.
A CoA requirement for the acetyl-CoA-primed reaction of chicken liver fatty acid synthetase is unequivocally established in conventional assays (Fig. 4) and stopped flow experiments (Fig. 5 ) by the use of a CoA trap. The small increase (rather than decrease) of lag period by added CoA in the stopped flow experiment using limited acetyl-coA (Table I, Experiment 12 versus 3) indicates that this requirement is 140 Transient Kinetic Studies of Fatty Acid Synthetase normally met by CoA released through loading of thioester substrates and that increased CoA preferentially stimulates unloading of enzyme-bound acetyl, rather than malonyl, residues as the result of differences in kinetic parameters of the two deacylation reactions. The larger increase in steady state cycle time and the inhibition of the steady state rate in conventional assays by CoA (cf. "Effects of CoA on Fatty Acid Synthesis from Acetyl-and Malonyl-CoA") are due to this effect and more importantly to inhibition of noncovalent association (Step 1 and Reaction 1 in forward direction) of both substrates. At high acetyl-coA (Fig. 6), this substrate is more extensively loaded. 10 ~L M CoA promotes its unloading, and this effect is manifested as a lag due to removal of competently bound acetyl residues obtained through Step 1 and a decrease in the steady state cycle time (Fig. 6, curve 2, and Table I, Experiment 13 versus 7) due to removal of abortive acetyl residues obtained at steps (3, 9, etc.) where malonyl-CoA loads. At 100 p~ CoA, the synthetase reaction is strongly inhibited (Fig. 6, curue 3, and Table I, Experiment 14) by inhibition of nonvalent association and unloading of both substrates. Additional CoA effects, such as those on the termination reaction (1,2 ) or reductase reaction, however, cannot be excluded.
The kinetic self-editing mechanism depicted in this study is similar to the proof-reading process in DNA polymerases and amino acyl-tRNA synthetases and provides fatty acid synthetase with the ability to correct errors in the loading of thioester substrates. For a more detailed understanding of this mechanism, equilibrium isotope exchange experiments are currently being carried out to determine the kinetic parameters of the loading reactions (Reaction l) for acetyl-and malonyl-CoA.