Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

PERGAMON The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 The International Journal of Biochemistry & Cell Biology Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate Yan Shi a, Wei Luo b, Wei-Xi Tian a, Tong Zhang b, Hai-Meng Zhou b, * a Graduate School, University of Science and Technology of China, Beijing 100039, People's Republic of China b Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, People's Republic of China Received 20 May 1998; received in revised form 19 August 1998; accepted 21 August 1998 Abstract Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mm) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Signi cant conformational changes (as indicated by the changes of the intrinsic uorescence emission and the ultraviolet di erence spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no signi cant e ect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational exibility than the enzyme molecule as a whole. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Fatty acid synthase; Chicken liver; Conformational changes; Sodium dodecyl sulfate; Inactivation 1. Introduction Fatty acids are essential components of all biological membranes and represent an important form of energy storage in both animals and Abbreviations: FAS, fatty acid synthase, ACP, acyl carrier protein, SDS, sodium dodecyl sulfate, NADPH, nicotinamide adenine dinucleotide phosphate, CoA, coenzyme A. * Corresponding author. Fax: +86-10-6256-8182; e-mail: zhm-dbs@mail.tsinghua.edu.cn. plants, so their biosynthesis occurs in all living organisms. The synthesis of fatty acids by fatty acid synthase [acyl-coa: malonyl-coa C-acyl transferase (decarboxylating, oxoacyl- and enoylreducing and thioester-hydrolyzing), EC 2.3.1.85] includes seven enzymatic activities. The animal fatty acid synthase comprises two multifunctional polypeptide chains, each containing seven discrete functional domains, juxtaposed head-to-tail so that two separate centers for fatty acid assembly are formed at the subunit interface [1]. The 1357-2725/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved. PII: S1357-2725(98)00101-0

1320 Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 fatty acid synthase from chicken liver is a dimeric molecule composed of identical, multifunctional subunits each with a molecular weight of 250,000 [2, 3]. Witkowski et al. [4] explored the structural organization of the individual component activities of the multifunctional animal fatty acid synthase. However, there have been few studies of the relationship between the inactivation of the overall reaction and inactivation of each component of the enzyme reaction in denaturant solutions as well as comparison of the inactivation of the enzyme and conformational changes of the whole enzyme molecule during denaturation. It has previously been reported that during the denaturation of a number of enzymes by guanidinium chloride or urea, inactivation occurs before noticeable conformational changes of the enzyme molecule as a whole can be detected [5±9]. Therefore, Tsou [10, 11] suggested that enzyme active sites are formed by relatively weak molecular interactions and, hence, may be conformationally more exible than the intact enzymes. The present investigation studies the inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver. The results show that denaturation of this multifunctional enzyme by SDS occurred in three stages. Comparison of inactivation and conformational changes of the enzyme suggests that the active sites of this multifunctional enzyme also display more conformational exibility than the enzyme molecule as a whole. with the following extinction coe cients: Chicken liver fatty acid synthase, 4.83 10 5 M 1 cm 1 at 279 nm; acetyl-coa, 1.54 10 4 M 1 cm 1 at 259 nm, ph 7.0; malonyl-coa, 1.46 10 4 M 1 cm 1 at 260 nm, ph 6.0; NADPH, 6.02 10 3 M 1 cm 1 at 340 nm and 1.59 10 4 M 1 cm 1 at 259 nm, ph 9.0; acetoacetyl-coa, 1.59 10 4 M 1 cm 1 at 259 nm, ph 7.0 [12]. Fatty acid synthase activity (for the overall reaction) was determined with a Perkin-Elmer Lambda Biospectrophotometer at 378C by following the decreasing of NADPH at 340 nm. The reaction mixture contained sodium phosphate bu er, 100 mm, ph 7.0; EDTA, 1 mm; acetyl-coa, 3 mm; malonyl-coa, 10 mm; 2. Materials and methods The preparation, storage and use of fatty acid synthase from chicken liver were as described previously in Ref. [12]. The preparation was homogeneous on polyacrylamide gel electrophoresis (PAGE) in the presence and absence of SDS. Acetyl-CoA, malonyl-coa, NADPH, acetoacetyl-coa and SDS were obtained from Sigma. All other reagents were local products of analytical grade. The concentrations of the enzyme and reagents were determined by absorption measurements Fig. 1. Inhibition of chicken liver fatty acid synthase by di erent concentrations of SDS. The reaction mixture contained di erent concentrations of SDS. The remaining activity was measured after the enzyme was mixed with the reaction mixture. Curves 1±4 represent the inactivations for the overall reaction of FAS (w), the reaction of acetoacetyl-coa reductase (q), b-ketoacyl reductase (r) and enoyl reductase (.), respectively.

Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 1321 Fig. 2. Lineweaver±Burk plots for the inhibition of chicken liver fatty acid synthase by SDS. Experimental condition was as for Fig. 1 except for the substrate concentration. Fixed acetyl-coa of 2.55 mm and NADPH of 30 mm, SDS concentrations of line 1±4 are 0, 0.053, 0.07 and 0.088 mm, respectively. NADPH, 35 mm and chicken liver fatty acid synthase in a volume of 1.0 ml. b-ketoacyl reductase activity was determined at 378C by measuring the change of absorption at 340 nm [13, 14]. The reaction mixture contained ethyl acetylacetate, 40 mm; NADPH, 35 mm; 1 mm EDTA and the enzyme in 100 mm phosphate bu er, ph 7.0. Enoyl reductase activity was also determined at 378C by measuring the change of absorption at 340 nm [13, 14]. The assay system contained 10 mm phosphate bu er, ph 6.3, 40 mm ethyl butenoate, 35 mm NADPH and 1 mm EDTA. Acetoacetyl-CoA reduction includes the four reactions transacylation, b- ketoacyl reduction, dehydration and enoyl reduction which are catalyzed by four component enzymes and ACP in fatty acid synthase. The initial rate of acetoacetyl-coa reduction was also determined at 378C by measuring the change of absorption at 340 nm [15]. The reaction mixture consisted of 10 mm phosphate bu er, ph 7.8 containing 20 mm acetoacetyl-coa, 35 mm NADPH and the enzyme. Ultraviolet di erence spectra were measured with a Perkin-Elmer Lambda Biospectrophotometer. A cuvette containing the enzyme dissolved in 100 mm phosphate bu er, ph 7.0 (con- Table 1 Kinetic parameters and inhibition constants of chicken liver fatty acid synthase inhibited by SDS Substrate Malonyl-CoA Acetyl-CoA NADPH K m (mm) 5.00 0.43 7.69 K i (mm) 0.14 0.14 0.15

1322 Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 taining 1 mm EDTA) was placed in the reference beam while another cuvette containing the same enzyme solution was placed in the sample beam. Di erent volumes of SDS (175 mm SDS) were added to the sample cuvette, and the same volumes of bu er were added to the reference cuvette. The spectra were recorded after mixing and incubation for 10 min at room temperature. Fluorescence emission spectra were measured using a Hitachi 850 spectro uorimeter. The excitation wavelength was 280 nm. Circular dichromism spectra were recorded on a Jasco 500C spectropolarimeter at room temperature. The sample cell volume was 200 ml with a path length of 0.1 mm. Four scans between 190 and 240 nm were successively added to ensure a good signalto-noise ratio. All measurements except the assay were carried out at 258C. 3. Results 3.1. Inactivation of fatty acid synthase in SDS solutions The e ect of SDS concentration in the 100 mm phosphate bu er (ph 7.0) on the inactivation of fatty acid synthase is shown in Fig. 1. The results show that at low concentrations of SDS, less than 0.15 mm, the activity for the overall FAS reaction was completely lost; however, both the b-ketoacyl reductase component and the enoyl reductase component still retained their complete initial activities. This result indicated that the FAS had been inactivated before inactivation at the active sites of both the b-ketoacyl reductase and enoyl reductase. Two possibilities have been considered for the above results. First, the loss of overall Fig. 3. Ultraviolet di erence spectra of chicken liver fatty acid synthase in various SDS solutions. Di erent volumes of SDS solutions were added to the sample cuvette with the same volumes of bu er being added to the reference cuvette. The enzyme concentration was 2 mm. The spectra were recorded after mixing and incubation for 10 min. The nal concentrations of SDS for curves 0±5 were 0, 0.35, 0.70, 1.05, 1.40 and 3.5 mm, respectively.

Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 1323 reaction activity of FAS may have been caused by rapid dissociation of the active enzyme dimer at low SDS concentrations into an inactive state. Secondly, small distance changes between the active sites of the component enzymes, not detectable by the methods Fig. 4. Fluorescence emission spectra of chicken liver fatty acid synthase denatured by SDS. Experimental conditions were as for Fig. 3. except that the enzyme concentration was 0.4 mm. The nal concentrations of SDS for curves 0±5 were 0, 0.35, 0.7, 1.4, 2.1 and 3.5 mm, respectively.

1324 Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 employed in this study, may have brought about the inactivation of FAS. The results of PAGE in the presence of 0.2 mm SDS show the enzyme dimer is no or little dissociation at low concentrations of SDS (electrophoresis data not shown). Therefore, the possibility that the inactivation of FAS is due to rapid dissociation of the active dimer into the inactive monomer can be excluded. even at high SDS concentrations. Control experiments showed that SDS had very little e ect on the intensity of the model compound, N-acetyl-Ltryptophan. 3.2. Inhibition type and kinetics parameters of fatty acid synthase by SDS FAS activity in the presence of various SDS concentrations was studied by changing concentrations of acetyl-coa, malonyl-coa and NADPH, respectively. Fig. 2 shows the Lineweaver±Burk plots for xed acetyl-coa and NADPH concentrations. The results show that SDS is a noncompetitive inhibitor for malonyl- CoA. The Lineweaver±Burk plots for xed malonyl-coa and NADPH concentrations and for the xed acetyl-coa and malonyl-coa concentration show that SDS is also a noncompetitive inhibitor for acetyl-coa and NADPH (data not shown), respectively. K m and K i can be obtained from the intercept of the straight lines on the 1/[S] axis and from suitable secondary plots, respectively. The obtained values of kinetic parameters were summarized in Table 1. 3.3. Conformational changes of fatty acid synthase in SDS solutions The ultraviolet di erence spectra for chicken liver fatty acid synthase in di erent concentrations of SDS are shown in Fig. 3. It can be seen that the denatured spectra minus the reference spectra showed two negative peaks at 287 and 294 nm. As the SDS concentration increased, the magnitude of the two negative peaks reached maximum values at 3.5 mm SDS. Figure 4 shows the uorescence emission spectra for chicken liver fatty acid synthase denatured in di erent SDS concentrations. As the SDS concentration increased, the uorescence emission intensity decreased in magnitude to nal values at 3.5 mm SDS, but no red shift was observed Fig. 5. CD spectra of chicken liver fatty acid synthase during denaturation by SDS. Experimental conditions were as for Fig. 3 except that the enzyme concentration was 1.0 mm. The nal concentrations of SDS for curves 0±6 were 0, 0.35, 0.70, 1.05, 2.10, 3.50 and 8.00 mm, respectively. Curve 7 represents the CD spectra of fully denatured enzyme in 7.2 M guanidinium chloride.

Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 1325 Figure 5 compares the CD spectra of the enzyme in di erent concentrations of SDS. As a control, curve 7 gives the CD spectrum of the enzyme in 7.2 M guanidinium chloride when the enzyme molecules were fully unfolded or disordered. For the di erent SDS concentrations, the nearly similar CD spectra indicate that the presence of SDS does not appreciably a ect the secondary structure of chicken liver FAS. Even at 8 mm SDS concentration (critical micelle concentration), the CD spectrum show no signi cant change. However, it is interesting that FAS may still retain some ordered structure in 7.2 M guanidine solution, as the curve 7 in Fig. 5 shows a small peak at 212 nm, rather than at 198 nm. The similar results were also observed during denaturation of reduced ribonuclease A in 6 M guanidine solution [16, 17]. 3.4. Comparison of inactivation and conformational changes of chicken liver fatty acid synthase The enzyme solutions were incubated with di erent concentrations of SDS for 10 min before determination of the remaining activity. The inactivation and conformational changes of chicken liver FAS during SDS denaturation are compared in Fig. 6. At low concentrations, less than 0.15 mm, the activity for the overall reaction of chicken liver FAS was completely lost, while no signi cant inactivation of the enzyme b-ketoacyl reductase component was observed. With further increases of the SDS concentration, the component enzymes were inactivated, while no observed conformational changes of the enzyme molecule. Further increases in the SDS concentration caused the enzyme to nally unfold. Fig. 6. Comparison of inactivation and conformational changes of chicken liver fatty acid synthase during denaturation by SDS. The enzyme solutions were incubated with SDS for 10 min before determination of the remaining activity of overall reaction of FAS (r), the remaining activity of the componental b-ketoacyl reductase of FAS (w), the changes of uorescence emission intensity at 334 nm (R) and the changes of di erence absorption at 287 nm (*).

1326 Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 Fig. 7. A semilogarithmic plot for the inactivation course of chicken liver FAS in 0.3 mm SDS solution. (w) Experimental data; (*) points obtained by subtracting the condition of the slow phase from curve (w). 3.5. Comparison of the rate constants of inactivation and conformational changes of fatty acid synthase in SDS solutions The enzyme was incubated at 258C with 0.3 mm SDS in 100 mm phosphate bu er, ph 7.0. 10 ml aliquots were taken at di erent time intervals and added to the assay mixture to measure the activity of overall reaction of FAS. Fig. 7 shows a semilogarithmic plot of the remaining activity versus time during inactivation by 0.3 mm SDS. The results show that the inactivation process consists of two rst-order reactions. The kinetic constants obtained from Fig. 7 are shown in Table 2. Similarly, the inactivation process of the b-ketoacyl reductase component in 0.5 mm SDS solution also consists of two rst-order reactions (Fig. 8). The kinetic constants are also summarized in Table 2. The uorescence emission spectra of chicken liver FAS at di erent time intervals were measured after the enzyme was mixed with 0.5 mm SDS. A semilogarithmic plot of the uorescence intensity at 334 nm versus time gives a curve which can be resolved into two straight lines, indicating that, like the inactivation

Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 1327 Table 2 Rate constants of inactivation and unfolding of chicken liver fatty acid synthase inhibited by SDS Inactivation (10 3 s 1 ) Unfolding (10 3 s 1 ) Overall reaction b-ketoacyl reduction Fluorescence change Concentration of SDS (mm) k 1 k 2 k 1 k 2 k 1 k 2 0.3 36.00 4.50 0.5 b b a a a a 23.00 c 1.2 c 19.00 0.25 bd bd a No signi cant change. b Reaction rate was too fast to be measured by conventional dynamic method. c and d represent the substrate systems in absence and presence of SDS of corresponding concentrations, respectively.k 1 and k 2 are the rst-order rate constants for the fast and slow phases, respectively. Fig. 8. A semilogarithmic plot for the inactivation of the b-ketoacyl reductase component of FAS in 0.5 mm SDS solution. (r) Experimental data; (R) points obtained by subtracting the condition of the slow phase from curve (r).

1328 Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 Fig. 9. A semilogarithmic plot for the kinetic course of the uorescence emission intensity at 334 nm in 0.5 mm SDS solution. (w) Experimental data; (*) points obtained by subtracting the condition of the slow phase from curve (w). reactions, the unfolding process also consists of two rst-order reactions (Fig. 9). The kinetic constants obtained from Fig. 9 are summarized in Table 2. 4. Discussion The present results show that much lower concentrations of SDS are required to bring about inactivation of the overall reaction of chicken liver FAS than are required to produce inactivation of the component enzymes, indicating that inactivation of the overall reaction of FAS is not due to changes of the active site structure of the component enzymes. The above results may be either due to the rapid dissociation of the active enzyme dimer into inactive monomers or due to small distance changes between the component enzyme active sites. It is well known that dissociation of the native enzyme into monomers results in the loss of FAS activity [18]. The results from PAGE in the presence of 0.2 mm SDS show that the dimer has little or no dissociation when the enzyme was inactivated. Therefore, the possibility that dissociation results in inactivation of the enzyme can be excluded. The most likely explanation is that a small distance change between the active site of the component enzymes results in inactivation of the chicken liver FAS. Although the crystal structure from X-ray analysis has not been reported [19], the changes in ultraviolet di erence spectra and in

Y. Shi et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1319±1330 1329 uorescence emission spectra observed in the present study re ect changes of the environments of Trp and Tyr residues of chicken liver FAS during unfolding by SDS. It has been previously reported that during the denaturation of creatine kinase by guanidine hydrochloride or urea, comparison of the activity changes with the extent of unfolding of the molecule shows that much lower concentrations of urea or guanidine are required to bring about inactivation than for signi cant conformational changes of the enzyme molecule. At the same concentrations of urea or guanidine, the inactivation rate constants of the enzyme are several orders of magnitude faster than the rate constants of conformational changes. Similar results have been obtained during denaturation of other enzymes. Comparing the unfolding and inactivation of several enzymes in the presence of urea and guanidinium chloride [5±9], Tsou [10, 11] suggested that active sites are usually situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole. Recently, we provided direct evidence relating the conformational change during guanidinium chloride denaturation and the exibility of the active site of creatine kinase [20]. However, previous authors have largely concentrated on studies of single function enzymes. Comparison of inactivation and conformational changes of multifunction enzymes has not been well explored. The present investigation compares the inactivation and unfolding of the multifunction enzyme chicken liver fatty acid synthase during denaturation by SDS. The results show that much lower concentrations of SDS are required to bring about the inactivation of the component enzymes than are required to produce conformational changes of the enzyme molecule. Comparing the rate constants of inactivation of the component enzymes of FAS and the conformational change of the FAS molecule at the same concentration of SDS shows that the inactivation rates were faster than the rates of conformational changes of the enzyme. The above results suggest that the active sites of multifunctional chicken liver fatty acid synthase are also situated in limited regions that have more conformational exibility than the enzyme molecule as a whole. Acknowledgements The present investigation was supported by Grant 39670174 of the China Natural Science Foundation for W.X.T. References [1] S. Smith, The animal fatty acid synthase: one gene, one polypeptide, seven enzymes, FASEB J. 8 (1994) 1248±1259. [2] R.Y. Hsu, S.L. Yun, Stabilization and physicochemical properties of the fatty acid synthetase of chicken liver, Biochemistry 9 (1970) 239±245. [3] C.L. Tang, R.Y. Hsu, Fatty acid synthase of chicken liver. Reversible dissociation into two nonidentical subcomplexes of similar size, J. Biol. Chem. 247 (1974) 2689±2698. [4] A. Witkowski, V.S. Rangan, Z.I. Randhawa, C.M. Amy, S. Smith, Structural organization of the multifunctional animal fatty acid synthase, Eur. J. Biochem. 198 (1991) 571±579. [5] S.J. Liang, Y.Z. Lin, J.M. Zhou, C.L. Tsou, P.Q. Wu, Z.M. Zhou, Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride, Biochim. Biophys. Acta 1038 (1990) 240±246. [6] Y.Z. Ma, C.L. Tsou, Comparison of the activity and conformation changes of lactate dehydrogenase H 4 during denaturation by guanidinium chloride, Biochem. J. 277 (1991) 207±211. [7] L.Y. Chen, M. Tian, J.S. Du, M. Ju, The changes of circular dichroism and uorescence spectra, and the comparison with inactivation rates of angiotensin converting enzyme in guanidine solutions, Biochim. Biophys. Acta 1039 (1990) 61±66. [8] H.R. Wang, T. Zhang, H.M. Zhou, Comparison of inactivation and conformational changes of aminoacylase during guanidium chloride denaturation, Biochim. Biophys. Acta 1248 (1995) 97±106. [9] Q.X. Chen, W. Zhang, W.Z. Zhang, S.X. Yan, T. Zhang, H.M. Zhou, Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase, J. Protein Chem. 15 (4) (1996) 359±365. [10] C.L. Tsou, Location of the active sites of some enzymes in limited and exible molecular regions, Trends Biochem. Sci. 11 (10) (1986) 427±429. [11] C.L. Tsou, Conformational exibility of enzyme active sites, Science 262 (1993) 380±381.

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