Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate.

Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.     

Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.     

The mechanism of binding of L-serine to tryptophan synthase from Escherichia coli

The mechanism of binding of L-serine to tryptophan synthase, which is the initial phase of the catalytic mechanism, has been studied by steady-state and stopped-flow kinetic techniques. The dependence of three separable rate processes on the concentration of L-serine is compatible with four different enzyme-substrate complexes, one of which lies on a branch in the pathway. By use of L-serine deuterated at the alpha carbon, it is possible to assign the deprotonation of the external aldimine of L-serine with pyridoxal 5'-phosphate to the most rapid observable binding step. Measurements at two pH values show that the rate-determining step in the synthesis of L-tryptophan changes from release of L-tryptophan at the optimal pH of 7.6 to the binding of L-serine at pH 6.5. Measurements at pH 7.6 in the presence of the substrate analogue indolepropanol phosphate show that the stronger binding of L-serine is probably due to stabilization of the catalytically competent enzyme--L-serine complex. At pH 7.6 L-serine is bound far more slowly to the beta 2 subunit than to the alpha 2 beta 2 complex of tryptophan synthase and retains its alpha carbon proton. For the beta 2 subunit, the rate-determining step of tryptophan synthesis is deprotonation of bound L-serine. The effect of bound alpha subunit is to increase both the rate of deprotonation and beta-elimination, shifting the rate-limiting step to the release of L-tryptophan.     

Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.     

Circular dichroism and fluorescence studies on the binding of ligands to the alpha subunit of tryptophan synthase.

Binding to the alpha subunit of tryptophan synthase induces extrinsic Cotton effects in the substrates indole (IND), indoleglycerol phosphate (IGP), and D-glyceraldehyde-3-P (D-GAP) and in the inhibitor indolepropanol phosphate (IPP). These effects disappear when the enzyme is denatured in guanidinium chloride. The induced circular dichroism (CD) was used to determine the dissociation constant and the number of binding sites for IPP. The dissociation constant so determined is equal to 48 muM and is in good agreement with the value of 48 muM obtained by equilibrium dialysis. From the temperature dependence of the dissociation constant, a value of -2.8 kcal/mol for the binding enthalpy was obtained. The determination of dissociation constants by means of extrinsic Cotton effects is shown to be quite feasible. CD competition experiments with glycerol phosphate (GP) suggest that IPP binds bifunctionally to the enzyme: via its indole part and its phosphate group. Indolepropanol, which lacks the phosphate group, does not show an extrinsic Cotton effect. Since the induced CD is strongly dependent on the binding geometry, the close similarity between the induced spectra in IPP and IGP is additional evidence that IPP is a good substrate analog. Binding to the enzyme results in a blue shift of the IPP fluorescence emission maximum. The dissociation constant determined by fluorescence titration equals 46 muM and agrees well with the values determined by the other two methods. Previous biochemical and fast kinetic studies suggested the existence of multiple conformational states for the enzyme and of ligand-induced conformational changes. No evidence was found in the far-uv CD spectra for conformational changes upon binding of IND and D-GAP. For IPP a very small effect was observed.     

Immunochemical characterization of a functional site of (Na+,K+)-ATPase

Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.     

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.     

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).     

Isomerization of the muscarinic receptor . antagonist complex.

The mechanism of binding of two antagonists, 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, to the muscarinic receptor was studied. The pseudo-first order rate constant of association showed a hyperbolic dependence on the concentration of the antagonist(s) indicating that the interaction involves two equilibria. The first binding equilibrium is reached rapidly and is characterized by dissociation constants 2.7 +/- 0.4 nM and 6.7 +/- 2.5 nM in phosphate buffer (0.05 M, pH = 7.4) for 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, respectively. The first binding equilibrium is followed by a slower isomerization step of the receptor . antagonist complex. The equilibrium constants for the isomerization step of the complex for both ligands were about 0.15. The overall constant of binding obtained as the product of the above constants shows good agreement with the results of equilibrium binding studies.     

Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.     

The pH-dependent binding of NADH and subsequent enzyme isomerization of human liver beta 3 beta 3 alcohol dehydrogenase

Human class I beta 3 beta 3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The beta 3 subunit has a Cys substitution for Arg-369 (beta 369C) in the coenzyme-binding site of the beta1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant beta 369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 +/- 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pKa = 7.2 +/- 0.1), and coincided with Vmax values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pKa = 8.1 +/- 0. 2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with Vmax for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of beta 369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.     

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.     

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).     

Interactions of GroEL/GroES with a heterodimeric intermediate during alpha 2beta 2 assembly of mitochondrial branched-chain alpha-ketoacid dehydrogenase. cis capping of the native-like 86-kDa intermediate by GroES

We showed previously that the interaction of an alphabeta heterodimeric intermediate with GroEL/GroES is essential for efficient alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like alphabeta heterodimer. The alphabeta heterodimer, as an intact entity, was found to bind to GroEL at a 1:1 stoichiometry with a K(D) of 1.1 x 10(-)(7) m. The 1:1 molar ratio of the GroEL-alphabeta complex was confirmed by the ability of the complex to bind a stoichiometric amount of denatured lysozyme in the trans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-alphabeta complex in cis, despite the size of 86 kDa of the heterodimer (with a His(6) tag and a linker). Incubation of the GroEL-alphabeta complex with Mg-ATP, but not AMP-PNP, resulted in the release of alpha monomers. In the presence of Mg-ATP, the beta subunit was also released but was unable to assemble with the alpha subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the beta subunit (K(D) < 4.15 x 10(-9)m) than the alpha (K(D) = 1.6 x 10(-8)m) for GroEL. Incubation of the GroEL-alphabeta complex with Mg-ATP and GroES resulted in dissociation and discharge of both the alpha and beta subunits from GroEL. The beta subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the beta subunit competent for reassociation with the soluble alpha subunit to produce a new heterodimer. We propose that this mechanism is responsible for the iterative annealing of the kinetically trapped heterodimeric intermediate, leading to an efficient alpha(2)beta(2) assembly of human branched-chain alpha-ketoacid dehydrogenase.