Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer<-->structured monomer) and unfolding (structured monomer<-->unfolded monomer) reaction steps, with the apparent transition midpoints (C(m)), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (-DeltaG(unf, aq)), is 4.4-5.5 kcal mol(-1), and that of native tetramer relative to structured monomer (-DeltaG(dis, aq)) is 7.2-7.4 kcal mol(-1), giving an overall free energy of stabilization (-DeltaG(dis&unf, aq)) of 11.6-12.9 kcal mol(-1) (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.     

Kinetic analysis of subunit oligomerization of the legume lectin soybean agglutinin

The reconstitution of soybean agglutinin (SBA), a tetrameric GalNAc/Gal-specific legume lectin, after denaturation in urea has been studied using fluorescence, far-UV CD, a hemagglutination assay, and chemical cross-linking with glutaraldehyde as a bifunctional reagent. The reconstituted protein exhibits similar quaternary structure and activity as of native lectin. The kinetics of subunit oligomerization has been determined from the cross-linking reaction of the reconstituting protein followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Monomers and tetramers could be quantitatively analyzed during reconstitution. Dimers are not detectable. The reassociation reaction follows second-order kinetics. The results are described by a kinetic mechanism in which the monomer-to-dimer association (characterized by a second-order rate constant (k(1)) of 1.4 x 10(4) M(-1) s(-1) at 37 degrees C) is involved in the rate-determining step of the oligomerization reaction.     

Denaturation-renaturation of the fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein

The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.     

Effects of low concentrations of guanidine . HCl on the reconstitution of lactic dehydrogenase from pig muscle in vitro. Evidence for guanidine binding to the native enzyme

The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.     

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.     

Mechanism and specificity of reconstitution of dimeric lactate dehydrogenase from Limulus polyphemus

D-Lactate dehydrogenase (EC 1.1.1.28) from Limulus polyphemus is a homodimer which is composed of identical subunits of Mr = 35 000. The enzyme may be reversibly denatured and dissociated at acid pH or in 6M guanidine X HCl. The sigmoidal time course of reactivation obeys a consecutive uni-bimolecular mechanism with k1 = 6 X 10(-4) S-1 and k2 = 1.3 X 10(-4) M-1 S-1 (20 degrees C) as first- and second-order rate constants. Cross-linking experiments with glutaraldehyde prove that reactivation and dimer formation run parallel. Joint "synchronous" reconstitution of the enzyme with dimeric porcine mitochondrial malate dehydrogenase (after denaturation in 6M guanidine X HCl) does not yield active hybrids. The unchanged kinetics of reactivation in the absence and presence of the prospective partner of hybridization prove that inactive hybrid intermediates may also be excluded. The absence of hybrids upon synchronous reconstitution of the two closely related dimeric NAD-dependent dehydrogenases clearly suggests that the assembly of nascent oligomeric proteins must be highly specific.     

Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM.

The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrading Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hydroxylase activity, whereas an inactive form accumulated in a recombinant strain. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by electrospray mass spectrometry, but nondenaturing gel filtration showed molecular masses of 31 600 Da for the inactive form and 11 500 Da for the active form. Cross-linking and sedimentation velocity results were consistent with the inactive form being a dimer. Partial thermal or chemical denaturation, or treatment with trifluoroethanol, readily activated dimeric DmpM. A combination of circular dichroism and fluorescence spectroscopies, activity assays, and native and urea gel electrophoresis were used to further characterize reactivation with urea. These results showed that dissociation of the dimeric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissociation also converts the monomeric form to a different conformation.     

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.     

Evidence for active intermediates during the reconstitution of yeast phosphoglycerate mutase

The reconstitution of the tetrameric phosphoglycerate mutase from bakers' yeast after denaturation in guanidine hydrochloride has been studied. When assays are performed in the presence of trypsin, it is found that reactivation parallels the regain of tetrameric structure. However, in the absence of trypsin, the regain of activity is more rapid, suggesting that monomeric and dimeric intermediates possess partial activity (35% of the value of native enzyme) which is sensitive to trypsin. When reconstitution is studied in the presence of substrates, it is again found that monomeric and dimeric intermediates possess 35% activity. Under these latter conditions, the activity of the monomer but not of the dimer is sensitive to trypsin.     

Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopical methods, fluorescence and circular dichroism. Three successive processes can be observed during the renaturation of this protein. First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer. Second, two monomers associate into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of ATP during renaturation increases the rate of formation of this dimeric species. The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site. The self-assembly of phosphofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.     

Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Comparison of the conformation and stability of the native dimeric,monomelic, tetrameric and the desensitized forms of the nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) fromPhaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without an alteration of the molecular weight, was achieved either by reversible denaturation with 6 M urea or by passage through a column of Blue Sepharose but additionofp-hydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the helical content, EDTA inactivation and Zn²⁺ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion. Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are operative in the desensitization of regulatory proteins.     

Renaturation and urea-induced denaturation of 20 beta-hydroxysteroid dehydrogenase studied in solution and in the immobilized state

Tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) from Streptomyces hydrogenans was reactivated after inactivation, dissociation and denaturation with urea. The effect of several factors such as NAD+, NADH, substrate, sulphydryl reducing agents, extraneous proteins, pH and enzyme concentration on reactivation was investigated. The coenzymes were found to be essential for obtaining a high reactivation yield (about 90%), since in their absence the reactivation was less than 10%. NADH was effective at lower concentrations than NAD+. The reactivated enzyme, after the removal of inactive aggregates, showed physical and catalytic properties coincident with those of the native enzyme. The mechanism by which NADH affects the reconstitution of 20 beta-hydroxysteroid dehydrogenase was investigated using both soluble enzyme and enzyme immobilized on Sepharose 4B. The immobilization demonstrates that isolated subunits are inactive and incapable of binding NADH and suggests that subunit association to the tetramer is essential for enzymatic activity. NADH appears to act, after subunit assembly has taken place, by stabilizing tetramers and preventing their aggregation with monomers that would give rise to inactive polymers.     

Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or with Pi in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of Pi binding and enzyme phosphorylation with Pi (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the Pi and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of Pi and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.     

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.     

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.     

Consequences of a six residual deletion from the N-terminal of rabbit muscle creatine kinase

A mutant of dimeric rabbit muscle creatine kinase (CK), in which six residues (residues 2-7) at the N-terminal were removed by the PCR method, was studied to assess the role of these residues in dimer cohesion and to determine the structural stability of the protein. The specific activity of the mutant was 70.39% of that of the wild-type CK, and the affinity for Mg-ATP and CK substrates was slightly reduced compared with the wild-type protein. The structural stability of the mutant was investigated by a comparative equilibrium urea denaturation study and a thermal denaturation study. The data acquired by intrinsic fluorescence and far-UV circular dichroism (CD) during urea unfolding indicated that, the secondary and tertiary structures of the mutant were more stable than those of wild-type CK. Furthermore, results of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence demonstrated that the hydrophobic surface of the mutant CKND(6) was more stable during urea titration. Data from size exclusion chromatography (SEC) experiments indicated that deletion of the six N-terminal residues resulted in a relatively loose molecular structure, but the dissociation of the mutant CKND(6) occurred later during the unfolding process than for wild-type CK. Consistent with this result, the differential scanning calorimetry (DSC) profiles demonstrated that the thermal stability of the enzyme was increased by removal of the six N-terminal residues. We conclude that a more stable quaternary structure was obtained by deletion of the six residues from the N-terminal of wild-type CK.     

Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD

Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.