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).     

Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding

Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPgammaS, binding of ATPgammaS and ADP to GroEL in the absence of ATP, and binding of ATP as monitored by fluorescence labelling. Thus, for the first time, multiple data sets for the interaction of nucleotides with GroEL are described quantitatively by an allosteric model. A noteworthy feature of our model is that no negative cooperativity in ATP binding occurs in accordance to experimental observations. Furthermore, the model also includes the existence of a conformation with very high ATPase activity. Such a conformation might be of importance at a certain stage in the folding cycle.     

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.     

Kinetic analysis of ATP-dependent inter-ring communication in GroEL

Different concentrations of ATP were mixed rapidly with single-ring or double-ring forms of GroEL containing the Phe44-->Trp mutation and the time-resolved changes in fluorescence emission, upon excitation at 295 nm, were followed. Two kinetic phases that were previously found for double-ring GroEL are also observed in the case of the single-ring version: (i) a fast phase with a relatively large amplitude that is associated with the T-->R allosteric transition; (ii) and a slow phase with a smaller amplitude that is associated with ATP hydrolysis. In the case of weak intra-ring positive cooperativity, the rate constant corresponding to the T-->R allosteric switch of single-ring GroEL displays a bi-sigmoidal dependence on ATP concentration that may reflect parallel pathways of the allosteric transition. The slow phase is absent when double-ring or single-ring forms of GroEL are mixed with ADP or ATP without K(+), and it has a rate constant that is independent of ATP concentration. A third fast phase that is still unassigned is observed for both single-ring and double-ring GroEL when a large amount of data is collected. Finally, a fourth phase is observed in the case of double-ring GroEL that is found to be absent in the case of single-ring GroEL. This phase is here assigned to inter-ring communication by (i) determining its dependence on ATP concentration and (ii) based on its absence from single-ring GroEL and the Arg13-->Gly, Ala126-->Val GroEL mutant, which is defective in inter-ring negative cooperativity. The value of the rate constant corresponding to this phase is found to increase with increasing intra-ring positive cooperativity, with respect to ATP. This is the first report of the rate of ATP-mediated inter-ring communication in GroEL, in the presence of ATP alone, which is crucial for the cycling of this molecular machine between different functional states.     

Different amino acid substitutions at the same position in the nucleotide-binding site of aspartate transcarbamoylase have diverse effects on the allosteric properties of the enzyme

Site-directed mutagenesis was used to determine how the allosteric properties of aspartate transcarbamoylase (ATCase) are affected by amino acid replacements in the nucleotide binding region of the regulatory polypeptide chains. Amino acid substitutions were made for both Lys-60 and Lys-94 in the regulatory chain since those residues have been implicated by x-ray diffraction studies, chemical modification experiments, and site-directed mutagenesis as playing a role in binding CTP and ATP. Lys-60 was replaced by His, Arg, Gln, and Ala, and Lys-94 was changed to His. These mutant forms of ATCase exhibit bewildering changes in the allosteric properties compared to the wild-type enzyme as well as altered affinities for the nucleotide effectors. The enzyme containing His-60 lacks both homotropic and heterotropic effects and exhibits no detectable binding of nucleotides. In contrast, the holoenzymes containing either Gln-60 or Arg-60 retain both homotropic and heterotropic effects. Replacement of Lys-60 by Ala yields a derivative exhibiting altered heterotropic effects involving insensitivity to CTP and activation by ATP. The mutant enzyme containing His-94 in place of Lys exhibits cooperativity with reduced affinity for nucleotides. The multiple substitutions at Lys-60 in the nucleotide binding region of the regulatory chains of ATCase demonstrate that different amino acids in the same location can alter indirectly the delicate balance of interactions responsible for the allosteric properties of ATCase. The studies show that it is hazardous and frequently unwarranted from single amino acid replacements of a specific residue to attribute to that residue the properties observed for the wild-type enzyme.     

Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.     

Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation

The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (T<-->R-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the reaction cycle, which implies that wild-type GroEL has evolved as a compromise between generality and specificity. We predict that, under maximum loading conditions and saturating ATP concentration, the efficiency of GroEL (using parameters for Rubisco) depends predominantly on the rate of R-->R" transition, while the equilibrium constant of the T<-->R has a small effect only. Both under sub- and superstoichiometric GroEL concentrations, enhanced efficiency is achieved by rapid turnover of the reaction cycle, which is in accord with the predictions of the iterative annealing mechanism. The effects are most dramatic at substoichiometric conditions (most relevant for in vivo situations) when SP aggregation can outcompete capture of SP by chaperonins.     

ATPase cycle of an archaeal chaperonin

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

Cooperativity in the thermosome

The thermosome from Thermoplasma acidophilum is a type II chaperonin composed of eight alpha and eight beta subunits. The genes encoding the two types of subunit were co-expressed in Escherichia coli and the alpha8/beta8 complex purified from the cell extract. The isolated complex showed steady-state ATPase properties characteristic of the thermosome purified from the native organism and was capable of enhancing the folding yield of a thermostable enzyme at elevated temperature (55 degrees C). To compare the nucleotide response of this double-ring structure with the type I and more compositionally heterogeneous type II chaperonins, the tryptophan residue within the alpha subunit was used as a fluorescence reporter of the conformational changes within the thermosome induced by the binding of nucleotides. Stopped-flow measurements of indole fluorescence at 55 degrees C showed that there is a fast (approximately 350 s(-1)) and a slow (approximately 0.6 s(-1)) structural rearrangement when ATP binds to the thermosome. Further examination of the fast rearrangement showed that the associated rate constant followed a two-phase saturation profile, as it does for GroEL and for the type II chaperonin from the eukaryotic cytoplasm. This result, in keeping with these precedents, reveals that the thermosome is also a negatively cooperative system with respect to inter-ring communications, i.e. the first ring loads with higher affinity than the second. As in the case of GroEL, the loading of the second ring is weakened by ADP, implying that asymmetric ATP/ADP complexes are favoured over symmetric ones. Despite the difference in co-protein involvement in the type I and II chaperonins, these observations show that negative cooperativity is a common feature of all chaperonins thus far examined. This property results in a strong preference for asymmetry in nucleotide occupancy and implies at least some commonality with the reciprocating encapsulation mechanism shown for the GroE chaperonins.     

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.     

A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding

Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.     

A dynamic model of long-range conformational adaptations triggered by nucleotide binding in GroEL-GroES

The molecular chaperone, GroEL, essential for correct protein folding in E. coli, is composed of 14 identical subunits organized in two interacting rings, each providing a folding chamber for non‐native substrate proteins. The oligomeric assembly shows positive cooperativity within each ring and negative cooperativity between the rings. Although it is well known that ATP and long‐range allosteric interactions drive the functional cycle of GroEL, an atomic resolution view of how ligand binding modulates conformational adaptations over long distances remains a major challenge. Moreover, little is known on the relation between equilibrium dynamics at physiological temperatures and the allosteric transitions in GroEL. Here we present multiple all‐atom molecular dynamics simulations of the GroEL‐GroES assemblies at different stages of the functional cycle. Combined with an extensive analysis of the complete set of experimentally available structures, principal component analysis and conformer plots, we provide an explicit evaluation of the accessible conformational space of unliganded GroEL. Our results suggest the presence of pre‐existing conformers at the equatorial domain level, and a shift of the conformational ensemble upon ATP‐binding. At the inter‐ring interface the simulations capture a remarkable offset motion of helix D triggered by ATP‐binding to the folding active ring. The reorientation of helix D, previously only observed upon GroES association, correlates with a change of the internal dynamics in the opposite ring. This work contributes to the understanding of the molecular mechanisms in GroEL and highlights the ability of all‐atom MD simulations to model long‐range structural changes and allosteric events in large systems. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.     

Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.     

Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Molecular chaperones are ATP‐consuming machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly. GroEL is an all‐purpose stochastic machine that assists misfolded substrate proteins to fold. The RNA chaperones such as CYT‐19, which are ATP‐consuming enzymes, help the folding of ribozymes that get trapped in metastable states for long times. GroEL does not interact with the folded proteins but CYT‐19 disrupts both the folded and misfolded ribozymes. The structures of GroEL and RNA chaperones are strikingly different. Despite these differences, the iterative annealing mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, T (apo), R (ATP bound), and R^″ (ADP bound). Analyses of the experimental data show that the efficiency of the GroEL–GroES machinery and mutants is determined by the resetting rate k _{R ″ → T} , which is largest for the wild‐type (WT) GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady‐state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.     

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co²⁺ and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a reflection that Gly-345 plays an essential role in conformational change and protein refolding by archaeal group II chaperonins.     

Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site

The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.     

Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis

The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis.