Substrate-induced conformational changes and half-the-sites reactivity in the Escherichia coli CoA transferase

ARCHIVES

OF

BIOCHEMISTRY

AND

Substrate-Induced Reactivity STEPHEN

525433

181,

Conformational in the Escherichia

J. SRAMEK,

Department

BIOPHYSICS

of Microbiology,

(1977)

Changes and Half-the-Sites co/i CoA Transferasel

FRANK E. FRERMAN, DANIEL GEORGE R. DUNCOMBE The Medical Received

College of Wisconsin, November

J. MCCORMICK,

Milwaukee,

Wisconsin

AND

53233

16, 1976

The acetyl-Cokacetoacetate CoA-transferase of Escherichia coli undergoes two detectable conformational changes during catalysis of CoA transfer. The first change occurs upon binding of at least the CoA moiety of an acyl-CoA substrate and was detected by fluorescence enhancement of enzyme-bound S-anilino-l-naphthalenesulfonate and microcomplement fixation upon formation of a noncovalent enzyme. CoA complex. CoA is a competitive inhibitor with respect to acyl-CoA substrate (Kj = 0.29 mM). A second, more extensive conformational change occurs upon formation of the covalent enzyme-CoA intermediate and was detected by fluorescence enhancement of enzymebound 8-anilino-1-naphthalenesulfonate, sedimentation of the intermediate in sucrose density gradients, and microcomplement fixation. The data clearly differentiated between the three distinct forms of the enzyme, i.e., free enzyme, noncovalent enzyme CoA complex, and covalent enzyme-CoA intermediate. The data are consistent with a model in which the enzyme opens upon formation of the enzyme-CoA intermediate. Either the limited conformational change or the extensive conformational change generates subunit interactions which result in half-the-sites reactivity in the enzyme. Only one of the two potential active sites was charged with etheno-CoA when the enzyme was reacted with etheno-acetyl-CoA. Glycerol abolished the extreme negative cooperativity and both active sites were charged with etheno-CoA in the presence of 10% glycerol. Our data suggest that glycerol abolished subunit interactions in either the enzyme. CoA complex or the covalent intermediate and not in the free enzyme.

Both the mammalian (1, 2) and Escherichia coli (3, 4) CoA transferases exhibit enhanced sulfhydryl group reactivity when the covalent enzyme-CoA intermediate is formed in the half reaction

mational changes in the catalytic pathway of the E. coli CoA transferase. Using acylCoA analogs, it was possible to demonstrate a limited conformational change when the CoA moiety binds to enzyme. A more extensive conformational change was demonstrated upon formation of the covalent intermediate. Our evidence suggests that one of these conformational changes induces negative cooperativity between the two active sites resulting in the half-the-sites reactivity.

0

II

R-C-SCoA

+ enzyme = enzyme-CoA

+ RCO,-.

In the case of the pig heart enzyme, this enhanced sulfhydryl group reactivity and accompanying loss of catalytic activity has been interpreted as a conformational change in which the enzyme opens upon formation of the covalent intermediate (1). In this communication, we present direct as well as indirect evidence for two confor-

EXPERIMENTAL

1 This research was supported by Grant AM 15527 from the National Institute of Arthritis, Metabolism, and Digestive Diseases and, in part, by a grant from the Wisconsin Heart Association.

PROCEDURES

Enzyme preparation. The E. coli CoA transferase was purified from E. coli CZ2 as previously described (3). Purification of the (I and /3 subunits of the CoA transferase was accomplished by isoelectric focusing of the urea-dissociated enzyme in a pH 3-10 gradient (supplemented with 0.2% pH 5-8 carrier ampholyte) in the presence of 6 M urea, 0.1 rnM dithiothreitol using an LKB 8101 column. The enzyme was exten-

525 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

SRAMEK

526

ET AL.

sively dialyzed against 6 M urea, 0.1 mM dithiothreito1 prior to focusing. Urea was treated with mixed bed resin and recrystallized prior to use. The peptides eluted from the column were passed through a column (1 x 90 cm) of Sephadex G-50 (fine) equilibrated with 0.05 M KPO,, pH 7.4, containing 0.1 mM dithiothreitol to remove the urea and ampholytes from the samples. The isolated subunits were identified based on their mobilities on polyacrylamide gel electrophoresis in dodecyl sulfate (3). This method was also used to assess the purity of the peptides. Based on densitometer scans of the stained gels, each subunit was at least 98% free of contamination by the other subunit. Full details of the purification will be published elsewhere.2 Enzyme assays. CoA transferase activity was routinely assayed by following the decrease in absorption at 232 nm due to the phosphotransacetylasecoupled arsenolysis of acetyl-CoA with butyryl-CoA and acetate as substrates (3). CoA transfer from acetyl-CoA to acetoacetate was assayed by coupling acetoacetyl-CoA formation to NADH oxidation in the presence of 3-hydroxyacyl-CoA dehydrogenase (4). Transfer of CoA from acetoacetyl-CoA to acetate was assayed by following the acetate-dependent disappearance of the Mg*+-enolate of acetoacetyl-CoA (3). Protein concentrations were determined by the Miller (5) modification of the procedure of Lowry et al. (6) with bovine serum albumin as the standard. The molar concentration of the CoA transferase was calculated based on a molecular weight of 98,000 (3). The molar concentrations of the a and p subunits were calculated based on molecular weights of 26,000 and 23,000, respectively (3). Sucrose gradient centrifugation. The CoA transferase was sedimented in 5-20% sucrose gradients and sedimentation coefficients were determined by the method of Martin and Ames (7). Gradients were formed from 5 and 20% sucrose solutions in 50 mM Tris (Cl-), pH 7.5, containing 0.1 mM dithiothreitol and 1 mM EDTA. Catalase, 11.3 S (8), and cytochrome c, 1.9 S (9), were used as internal standards. The proteins were layered on ll-ml gradients in 200 ~1 of the buffer containing 2% sucrose. Gradients were centrifuged for 16 h at 4°C in a No. 488 rotor on an International B-60 centrifuge at 40,000 rpm. Substrates were added to the 5 and 20% buffered sucrose solutions prior to forming some gradients as described in the text. The approximate molecular weight of the enzyme-CoA intermediate was calculated from the centrifugation data by the relationship (7)

using the free CoA transferase as the standard (3), where S is the sedimentation coefficient and M, is molecular weight. Fluorescence measurements. The fluorescence measurements were made with an Amino-Bowman spectrofluorometer equipped with a ratio photometer. Relative intensities were recorded directly as emission or excitation spectra either manually or with an X-Y recorder. In the experiments using ANS3 the dye was excited at 390 nm. The concentration of ANS in these experiments was 0.27 mM and the concentration of the CoA transferase was 16 WM. The concentration of ANS was determined spectrophotometrically at 406 nm using an extinction coefficient of 4.95 x lo3 M-I (10). The experiments were conducted in 0.05 mM Tris (SO,*-), pH 7.5, 1 mM EDTA, and 0.1 mM dithiothreitol. ANS had no effect on enzyme activity and substrates alone had no effect on ANS fluorescence. The concentration of c-CoA in the enzyme-CoA intermediate was determined fluorimetrically as previously described (11). The enzyme (7.2 nmol) was incubated with 0.5 mM l -acetyl-CoA for 2 min at 25°C in Tris (S0,2-), pH 7.5, 1 mM EDTA, and 0.1 mM dithiothreitol. The reaction mixture was then passed through a column (1 x 30 cm) of Sephadex G50 (fine) at 4°C. The column was eluted with 50 mM Tris (S0,2-), pH 7.4, containing 1 mM EDTA and 0.1 mM dithiothreitol. Protein, which eluted in the void volume of the column, was determined as described l -CoA was quantitated above and enzyme-bound fluorimetrically. When the enzyme:+CoA ratio was determined in the presence of glycerol, the incubation was conducted in 10% glycerol and the column was eluted with buffer containing 10% glycerol. The columns eluted with buffer containing glycerol were maintained at the same flow rate as those without glycerol by use of a peristaltic pump. The fluorescence yield of c-CoA was greater in the glycerolcontaining buffer; therefore, the relationship between relative fluorescence and l -CoA concentration (11) was also determined in the glycerol-containing buffer. Kinetic measurements. Kinetic data were analyzed using the FORTRAN programs of Cleland

s_1= M,, 213 ( Mm 1 ’ S,

3 Abbreviations used: ANS, 8-anilino-l-naphthalene sulfonate; e-CoA, 1-NG-etheno-CoA; e-acetylCoA, I-N”-etheno-acetyl-CoA, Nbs,, 5,5 -dithiobis(2nitrobenzoic acid); DEAE, diethylaminoethyl; IgG, immunoglobulin G, IgM, immunoglobulin M.

2 G. R. Duncombe script in preparation.

and F. E. Frerman,

manu-

(12). Difference spectra. Solvent perturbation difference spectra were recorded with a Cary 17D spectrophotometer at 25°C using the tandem cell arrangement of Herskowitz and Laskowski (13). N-Acetyltryptophan methyl ester was employed as the reference compound (14).

CONFORMATIONAL

CHANGES

Preparation and assay of anti-CoA transferase. Antisera to the CoA transferase were prepared in two New Zealand white rabbits. The rabbits were pre-bled for a source of nonimmune sera. The rabbits were inocula.Bd subcutaneously at multiple sites with 1 mg of CoA transferase in 1 ml of distilled water emulsified in 1 ml of Freund’s complete adjuvant. The rabbits were bled after 4 weeks, at which time anti-CoA transferase could be demonstrated in the sera from both rabbits by Ouchterlony doublediffusion analysis. Subsequent injections were made at 4 and 7 weeks as described above except that incomplete adjuvant was substituted. At 10 weeks, the rabbits were bled for a source of anti-CoA transferase. The crude immunoglobulin fraction was purified from the pooled antisera by ammonium sulfate fractionation (15) and chromatography on DEAE-cellulose (16). The purified anti-CoA transferase was characterized as an immunoglobulin by Ouchterlony double-diffusion analysis using goat anti-rabbit IgG and anti-IgM. The goat anti-globulins were the gifts of Dr. Jeffrey Winkelhake of this department. The rabbit anti-CoA transferase was assessed as pure IgG by immunoelectrophoresis using the goat anti-rabbit globulins. Using the purified CoA transferase subunits, only anti-a antibody could be detected by double-diffusion analysis (Fig. 1) and by microcomplement fixation (Fig. 2). The slight spur seen in Fig. 1 indicates that the anti-CoA transferase is directed primarily against the a subunit in the native state of the tetramer. The data illustrated in Figs. 1 and 2 show that the anti-CoA transferase

FIG. 1. Ouchterlony double-diffusion analysis of the specificity of anti-CoA transferase. One hundred microliters of the free enzyme, E (540 pg), of the purified a subunit (200 Kg), and of the purified /3 subunit (240 pg) were placed in the designated wells and allowed to diffuse for 16 h at 4°C. Anti-CoA transferase, AE (120 pg/lOO PI), was then added to the central well and diffusion was allowed to continue for an additional 16 h at 4°C.

IN E. coli CoA TRANSFERASE

527

FIG. 2. Microcomplement fixation patterns of CoA transferase and purified subunits using purifled anti-CoA transferase. Microcomplement fixation was performed as described by Levine and van Vunakis (18). Results are expressed as percentage complement fixed per picomole of purified subunit and per picomole of subunit in the tetramic enzyme. cross reacts with the a subunit and that most, if not all, of the antigenic determinants in the native enzyme are also present in the isolated a subunit. The lateral shift of the a curve (Fig. 2) also indicates that the conformation of the isolated LYsubunit is different from the a subunits in the native enzyme (17). The activity of the enzyme can be completely inhibited by the anti-CoA transferase. Ouchterlony double-diffusion analysis was conducted in 0.6% agarose in 0.05 M Tris-phosphate buffer, pH 7.4. Plates were run for 16 h at 4°C. Immunoelectrophoresis was performed in 1% ionagar on 8 x 11 plates in 0.025 M barbital buffer, pH 8.6, at 4°C. Samples were electrophoresed for 2 h at 7-12 mA at a constant voltage of 220 V. Microcomplement fixation was performed as described by Levine and van Vanukis (18). Materials. Acyl-CoA substrates, CoA, NADH, Clostridium kluyveri phosphotransacetylase, l-N6etheno-acetyl-CoA, and I-NG-etheno-CoA were obtamed from P-L Biochemicals. Cytochrome c (equine, type III), bovine liver catalase, and lithium acetoacetate were purchased from Sigma Chemical Co. N-Acetyl methyl esters of tyrosine and tryptophan and deuterium oxide (99.8 atom%) were obtained from Aldrich Chemical Co. Sephadex G-50 (fine) and G-100 were obtained from Pharmacia. Sheep blood was purchased from Baltimore Biological Laboratory. Guinea pig complement and rabbit anti-sheep erythrocyte stromata serum were purchased from Microbiological Associates. All other chemicals were purchased from commercial sources and were of the best grade available. RESULTS

Sedimentation of the enzyme4ToA compound. The increased sulfhydryl reactivity in the E. coli CoA transferase after incu-

bation with an acyl-CoA substrate sug-

SRAMEK ET AL.

528

gested that the enzyme undergoes a conformational change upon formation of the covalent enzyme-CoA intermediate (3, 4). Direct evidence for a conformational change upon formation of the covalent intermediate was obtained by velocity centrifugation of the CoA transferase in sucrose gradients containing acetoacetylCoA. When the enzyme was sedimented in the presence of substrates, either acetoacetate (100 mM = 10Ki) or acetoacetyl-CoA (0.26 lll~ = 1OKJ was added to the 5 and 20% buffered sucrose solutions prior to forming the gradients. Catalase and cytochrome c were applied to the gradients with the CoA transferase and served as internal standards. The native enzyme was always centrifuged in these experiments for comparison. Figure 3 illustrates the sedimentation behavior of the CoA transferase in the presence of acetoacetate (A) and the enzyme-CoA compound formed in the presence of acetoacetyl-CoA (B). In the presence of acetoacetate, the enzyme had a sedimentation coefficient of 5.36 S, which was not significantly different from that of the free enzyme (5.38 S) (3). In contrast, the enzyme-CoA intermediate had a sedimentation coefficient of

+.03

j.3 E

f.02 6 N.01 u

52 G .f .,

4.65 S. The decreased sedimentation coefficient is probably not the result of dissociation of the enzyme. Loss of a single subunit would yield a trimer with a molecular weight of about 75,000. Calculation of the approximate molecular weight of the enzyme-CoA intermediate from the experimentally determined sedimentation coefficient using the free enzyme as the standard yielded a molecular weight of 88,000. This apparent molecular weight is not consistent with a dissociation but rather reflects a change in the Stokes radius of the enzyme. In addition, gel filtration chromatography of the enzyme-CoA compound on a calibrated Sephadex G-100 column (1 x 50 cm) showed that neither enzyme activity nor protein was detectable in those fractions in which dimeric (a/?, (Ye,or &> or monomeric (a or /3>species would have been eluted. fmmunochemical studies. Evidence for distinct conformational states of the free enzyme and enzyme-CoA compound was also obtained by studying the inactivation of the free enzyme and enzyme-CoA compound by anti-CoA transferase. The free enzyme and enzyme-CoA compound (formed in the presence of 100 PM aceto-

i 5.010

,E &xx

a

l-L!& FIG. 3. Sedimentation of the CoA transferase and enzyme-CoA intermediate. The E. coli CoA transferase was sedimented in 5-20% sucrose gradients in the presence of 100 rnM acetoacetate (A) and 0.26 mM acetoacetyl-CoA (B) to form the enzyme-CoA intermediate. Catalase and CoA transferase were determined by catalytic activities and cytochrome c was determined by its absorbance at 412 nm. The direction of sedimentation was from right to left.

CONFORMATIONAL

acetyl-CoA) were incubated at 20°C for 1 min with anti-CoA transferase. Control incubations received either nonimmune sera (200 pg/lOO ~1) or no antisera. The concentration of the enzyme was 68 pg/lOO ~1 and the protein concentration of the anti-CoA transferase preparation was 50 pg/lOO ~1. The data in Table I show that the catalytic activity of the free enzyme was inhibited by about 50%, whereas the catalytic activity of the enzyme-CoA compound was inhibited by about 72% in the presence of the anti-CoA transferase. No difference in the level of inhibition was obtained when the anti-CoA transferase concentration was halved or doubled. The data were consistent with a conformational change of the enzyme resulting in an opening of the enzyme upon formation of the covalent intermediate and the exposure of additional antigenic sites. Additional data supporting distinct conformational states of the CoA transferase were obtained by examining the complement-fixing properties of the free enzyme, the enzyme-CoA intermediate, and the noncovalent complex, enzyme *CoA (Fig. 4). CoA is a competitive inhibitor with respect to acetoacetyl-CoA in the transfer of CoA from acetoacetyl-CoA to acetate. The apparent Ki for CoA was 0.29 f 0.03 TABLE I INHIBITION OF CATALYTIC ACTIWTIES OF THE CoA TRANSFERASE AND ENZYME-COA COMPOUND BY

ANTI-COA TRANSPERASE Addition Enzyme form”

CoA transferase

Enzyme-CoA termecliate

in-

529

CHANGES IN E. coli CoA TRANSFERASE

Percentage control activityb

Anti-CoA transferase Nonimmune serum

48 5 5

Anti-CoA transferase Nonimmune serum

28 f 2

97

101

a The free enzyme or enzyme-CoA intermediate generated in the presence of 100 PM acetoacetyl-CoA was treated for 1 min at 25°C with anti-CoA transferase or nonimmune serum in 50 rnM Tris (Cl-), pH 7.5, 1 mM EDTA. * Data are expressed relative to a control incubation that received no serum. The values represent the average and range of four experiments.

.l

.2

.3

CoA TRANSFEWE

.4 (,ug/ml

.5

.6

)

FIG. 4. Microcomplement fixation patterns of the CoA transferase, the covalent enzyme-CoA intermediate, and the noncovalent enzyme.CoA complex. Microcomplement fixation was performed as described by Levine and van Vunakis (18). The concentration of CoA transferase on the abscissa refers to concentration in the final 6-ml incubation. In these experiments, CoA transferase was incubated in l-ml incubations either alone or with 10 PM acetoacetyl-CoA to generate the covalent intermediate or with 5 mM CoA to generate the noncovalent complex; after about 30 s, 1 ml of anti-CoA transferase (0.75 mg/ml) was added and the mixture was diluted to a volume of 6 ml with diluter (18) and complement was added.

mM when acetoacetyl-CoA was varied from 15-110 PM and acetate was held constant at 30 mM. The complement fixation data shown in Fig. 4 indicate distinct conformations that are characteristic of each enzyme species. From its behavior as a competitive inhibitor with respect to acetoacetyl-CoA, CoA may be regarded as an acyl-CoA analog. These results are consistent with results of sulfhydryl modification experiments (4) in which the acetylCoA analog, acetylaminodesthio-CoA, enhanced sulthydryl group reactivity when incubated with the transferase and enhanced thiol group reactivity, presumably reflecting a conformational change in the Michaelis complex. Taken together, our data indicate two conformational changes in the catalytic pathway of CoA transfer: The first occurs upon binding of at least the CoA moiety of an acyl-CoA substrate and the second occurs upon formation of the covalent enzyme-CoA compound. The complement-fixing properties of the covalent enzyme-CoA compound (Fig. 4) are also consistent with an opening of the enzyme as well as an extensive conforma-

SRAMEK

530

tional change, reflected in the lateral shift of the curve, relative to that of the free enzyme (17). Binding zyme-CoA

of ANS to the enzyme and encompound. The CoA transfer-

ase binds the fluorescent dye, ANS. When the free dye was excited at 390 nm, the emission maximum was located at 500 nm. Upon binding to the CoA transferase, the emisison maximum shifted to 495 nm and a 30% increase in fluorescence was observed. The fluorescence was further enhanced upon addition of acetoacetyl-CoA (0.13 mM) to form the enzyme-CoA compound. There was no change in the wavelength of the emission maximum of the bound ANS upon formation of the enzymeCoA compound. Addition of carboxylic acid substrates did not enhance fluorescence. Table II shows the enhancement of enzyme-bound ANS fluorescence upon addition of substrates. Titrations with ANS were not carried out to determine whether the enhanced fluorescence resulted from a change in the fluorescence yield of bound dye or the binding of additional dye. Either interpretation is consistent with conformational change in the enzyme upon formation of the covalent intermediate. Free CoA (5 mM> also enhanced enzyme-bound dye fluorescence, consistent with the results of the complement-fixation studies, suggesting a limited conformational change. Effect of glycerol on CoA:enzyme stoichiometry. In the E. coli CoA transferase,

the fl subunit must contribute at least part of the active center since the glutamate residue to which CoA becomes covalently bound is located in the p subunit (4). The quaternary structure of the enzyme has been established as a& (3, 4); therefore, the enzyme contains two potential active TABLE II EFFECT OF SUBSTRATES ON THE FLUORESCENCE OF SANILINO-l-NAPHTHLENEBULFONATE BOUND TO CoA TRAN~FERA~E Addition (mM) Fluorescence enhancement (n-fold) Acetoacetyl-CoA (0.13) CoA (5) Acetoacetate (50) Acetate (50)

1.50 1.16 1.02 1.01

ET AL.

sites. Sulfhydryl groups reacted asymmetrically with Nbs, in the free CoA transferase in the presence and absence of 10% glycerol. That is, only one sulfhydryl reacted rapidly in this enzyme that has a symmetrical subunit composition, (Y&, and two potential active sites (3, 4). In contrast, two rapidly reacting sulfhydryls were observed in the enzyme-CoA compound in the presence of 10% glycerol (3). These data suggested that glycerol abolished subunit interactions in the enzymeCoA compound. It was hypothesized that the asymmetry and symmetry of sulfhydry1 group reactivity in the enzyme-CoA compound which was solvent dependent might also be reflected in the number of functional active sites. The stoichiometry of CoA bound per enzyme was therefore determined in the presence and absence of 10% glycerol. Enzyme-bound CoA was quantitated fluorometrically using e-acetyl-CoA, a fluorescent analog of acetylCoA which is a substrate of the bacterial enzyme (11..The data shown in Table III show that one active site was charged with E-CoA in the absence of glycerol, while two active sites were charged with l -CoA in the presence of glycerol. Thus, asymmetry of sulfhydryl reactivity (4) does reflect an asymmetry of active sites that results from modifier- or substrate-induced subunit interactions. Effect ofglycerol on kinetic contents and conformation of the native enzyme. The

results of the e-CoA binding experiments, the sulfhydryl modification experiments, and the sucrose velocity gradients demonstrated that the formation of the enzymeCoA intermediate resulted in an altered conformation of the CoA transferase. These experiments also indicated that the conformation of at least the covalent intermediate was perturbed by glycerol. A simple kinetic analysis was performed to determine if glycerol affected the kinetic constants of the reaction catalyzed by the CoA transferase. The results of the analysis are shown in Table IV. Initial velocities were determined in the presence of saturating acetoacetate and in the presence and absence of 10% glycerol. Both the apparent Michaelis constant for acetyl-CoA and the apparent maximum velocity were found to

CONFORMATIONAL TABLE

CHANGES

III

EFFECT OF GLYCEROL ON THE STOICHIOMETRY OF ECoA TO ENZYME IN THE COVALENT INTERMEDIATE Conditions”

Moles of c-CoA per mole of enzyme*

No glycerol 10% glycerol

1.10 + 0.20 1.90 + 0.05

a The enzyme was charged with e-CoA in the presence of 0.40 mM e-acetyl-CoA in 0.05 M Tris (SO,*-), pH 7.5, 0.1 mM dithiothreitol, 1 mM EDTA in the presence or absence of 10% glycerol. The reaction mixtures were passed through a column of Sephadex G-50 fine and covalently bound l -CoA and enzyme were quantitated as described under Experimental Procedures. * The values represent the average and range of values obtained in three experiments. TABLE

IV

EFFECT OF GLYCEROL ON THE KINETIC CONSTANTS OF THE E. coli CoA TRANSFERASE No glycerol 10% glycerol Constant” K,, acetyl-CoA (mM) V (~mol/min/unit) K,,IV

0.16 t 0.03 2.21 2 0.10 0.07

0.08 + 0.01 0.64 + 0.01 0.12

a Initial velocities were determined by coupling acetoacetyl-CoA formation to NADH oxidation in the presence of 3-hydroxybutyryl-CoA dehydrogenase (3). Kinetic constants and standard errors were determined as described under Experimental Procedures (12).

decrease in the presence of glycerol when compared to the values obtained in the absence of glycerol. Glycerol has little, if any, effect on sulfhydryl group reactivity in the free enzyme (4). Further evidence that glycerol did not affect the conformation of the native enzyme was obtained by examining the A 292.,lA zso nm ratio of the native enzyme relative to that of the model compound, N-acetyltryptophan methyl ester, as a function of glycerol concentration. Nacetyltryptophan methyl ester was used exclusively since the CoA transferase contains 42 tryptophan residues per mole and only 4 tyrosine residues (3). The data are illustrated in Fig. 5. The data show that glycerol has no detectable effect on the conformation of the free enzyme as judged by tryptophan perturbation and indicate that the effect of glycerol on the kinetic properties resulted from a perturbation of

531

IN E. coli CoA TRANSFERASE

the conformation of the enzyme-CoA intermediate or enzyme *acyl-CoA Michaelis complex. In addition, solvent perturbation spectra of the CoA transferase in 10% glycerol or 81% deuterium oxide indicated that 31 + 4% of the tryptophan residues were exposed to glycerol and 31 + 1% of the tryptophan residues were exposed to D,O and therefore presumably to solvent H,O. Therefore, with respect to glycerol or D%O, the same fraction of tryptophan residues is exposed to perturbants that differ by a factor of almost 3 in molecular radii:glycerol, 2.7 A; DzO, 1.0 A. DISCUSSION

A limited, acyl-Cob-induced conformational change and a general, extensive conformational change that occurs upon formation of the covalent intermediate of the E. coli CoA transferase were initially suggested by the results of sulfhydryl modification studies (3, 4). The data presented in this paper are consistent with the conclusions reached from the results of sulfhydry1 group modification data and permit qualitative interpretations regarding the conformational states of the enzyme during catalysis. Free CoA is a competitive inhibitor with respect to acyl-CoA substrate of the E. coli CoA transferase. Therefore, the noncovalent enzyme *CoA complex is a reasonable 1.5 t R

0

On

0

1.0 -

.5 -

I 10

I 20

% GLYCEROL FIG. 5. Effect of glycerol concentration on absorption of the CoA transferase relative to acetyl-tryptophan methyl ester. The absorbances 2.8 pM CoA transferase and 2.8 PM N-acetyl-tryptophan methyl ester (NAT) were determined at and 280 nm as a function of the concentration glycerol. The value plotted on the ordinate, R, is ratio (Aw,,.,/A%% .,,J/~AWn,,,/A~~~,,J.

the Nof 292 of the

532

SRAMEK

analog of an enzyme *acyl-CoA Michaelis complex. The complement fixation pattern of the enzyme *CoA complex is similar, but not identical to the pattern of the free enzyme. Less complement was fixed by the complex than the free enzyme indicating either that fewer sites in the complex are available for the reaction with anti-CoA transferase or that the binding affinity of those sites for anti-CoA transferase is lower while the majority of antigenic sites remain unchanged. These results parallel the results obtained when the noncovalent enzyme *acetylaminodesthiocomplex, CoA, was reacted with thiol reagents (4). In the latter complex, the rate but not the extent of sulfhydryl group reactivity was enhanced relative to free enzyme. In contrast, both the rate and the extent of sulfhydryl group reactivity increased in the covalent enzyme-CoA intermediate. The fluorescence enhancement data presented here qualitatively support these results and all data indicate a limited conformational change upon binding of the CoA moiety of an acyl-CoA substrate. The sedimentation properties of the covalent enzyme-CoA intermediate as well as the complement fixing properties of this form of the enzyme demonstrate a general, extensive conformational change when the covalent intermediate is formed. The opening of the enzyme is evident from the change in the sedimentation coefficient of the enzyme. The complement fixation data and inhibition by anti-CoA transferase provide a semiquantitative corroboration of that interpretation. The percentage complement fixed by the covalent intermediate and inhibition of catalytic activity of the covalent intermediate were greater, relative to the same concentration of enzyme, suggesting exposure of additional antigenic sites. Based on sulfhydryl reactivity data, White et al. (1) suggested that the pig heart succinyl-CoA:3-ketoacid CoA-transferase undergoes a conformational change upon formation of a covalent enzyme-CoA intermediate. They interpreted their data as an opening of the enzyme when the intermediate is formed. A general, extensive conformational change is apparently common in the reactions catalyzed by both the mammalian

ET AL.

and E. coli CoA transferases (l-4). The role of the conformational change is not clear. White and Jencks (19) have hypothesized that the CoA moiety of acyl-CoA substrates forces open the active site. However, the specific binding of acyl-CoA substrate is not reflected by the Gibbs’ free energy of binding. Rather, they suggested that this binding energy is utilized to destabilize the thiolester substrate or serve as the principal driving force for the extensive conformational change. Our data using free CoA and acyl-CoA analogs (4) strongly support the idea of a limited conformational change upon binding of the CoA moiety. The formation of the covalent intermediate probably contributes to rate enhancement. This idea can be rationalized if the enzyme functional group that participates in group transfer is a better nucleophile than the second substrate and a better leaving group than the leaving group on the first substrate (20). In the E. coli CoA the two conformational transferase, changes may render the y-carboxyl of glutamate at the active center, first, a better nucleophile than carboxylate substrate and, then, a better leaving group than the carboxylate product. The quaternary structure of the E. coli CoA transferase is a& (3, 4). The (Ysubunit contains the glutamate residue to which CoA is covalently bound during catalysis; thus, the enzyme contains two potential active sites per tetramer. Our data clearly show extreme negative cooperativity in the enzyme which results in the charging of only one active site per tetramer with E-CoA in the absence of glycerol. Glycerol abolished the negative cooperativity, permitting the charging of both active sites. The half-the-sites reactivity (21) in the E. coli CoA transferase undoubtedly results from subunit interactions which are induced by either binding of acyl-CoA substrate or formation of the covalent intermediate. It is not clear at this point which event gives rise to the observed negative cooperativity. Other data supporting subunit interactions have been obtained in experiments modifying the E. coli CoA transferase with pyridoxal S-phosphate (22) and thiol reagents (4). In the latter

CONFORMATIONAL

CHANGES IN E. coli CoA TRANSFERASE

case, the asymmetric reaction of sulfhydry1 group with thiol reagents was also abolished in the enzyme-CoA intermediate in the presence of glycerol. Glycerol has no significant effect on the structure of the free enzyme, as judged by sulfhydryl group reactivity and solvent perturbation of tryptophan. Its effects on the kinetic constants probably reflect a relaxation of subunit interactions after binding acylCoA or formation of the covalent enzymeCoA intermediate. Glycerol has previously been shown to affect subunit interactions in a mammary glucose 6-phosphate dehydrogenase (23), a bacterial aconitate isomerase (24), and phosphorylase b (25). REFERENCES 1. WHITE, H., SOLOMON, P., AND JENCKS, W. P. (1976) J. Biol. Chem. 251, 1700-1707. 2. FENSELAU, A., AND WALLIS, K. (1974). Biochemistry 13, 3884-3888. 3. SRAMEK, S. J., AND FRERMAN, F. E. (1975)Arch. Biochem. Biophys. 171, 14-26. 4. SRAMEK, S. J., FRERMAN, F. E., AND ADAMS, M. B. (1977) Arch. Biochem. Biophys. 181, 516

524. 5. MILLER, G. L. (1959)Anal. Chem. 31, 964. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193,

265-275. 7. MARTIN, R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 8. SUMMER, J. B.., AND GRALEN, N. (1938) J. Biol. Chem. 125, 33-36. 9. MARGOLIASK, E., AND LUSTCARTEN, J. (1962) J. Biol. Chem. 239, 3379-3405.

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