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Published online before print May 2, 2006, 10.1110/ps.062100606
Protein Science (2006), 15:1270-1276. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis

Oded Danziger1, Liat Shimon1 and Amnon Horovitz

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

(RECEIVED January 18, 2006; FINAL REVISION February 24, 2006; ACCEPTED March 6, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Conclusions
 Materials and methods
 Appendix
 References
 
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.

Keywords: chaperonins; molecular chaperones; allostery; cooperativity; protein folding


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Conclusions
 Materials and methods
 Appendix
 References
 
The GroE chaperonin system facilitates protein folding in an ATP-dependent manner (for reviews, see Horovitz et al. 2001; Thirumalai and Lorimer 2001; Saibil et al. 2002; Horovitz and Willison 2005). It consists of GroEL, an oligomer of 14 identical subunits of 57.3 kDa that form two stacked back-to-back heptameric rings (Braig et al. 1994) and its helper protein, GroES, which is a seven-membered ring of identical subunits of 10 kDa (Hunt et al. 1996). Each subunit of GroEL is made up of three domains: (1) an apical domain that binds GroES and nonfolded protein substrates, (2) an equatorial domain that contains an ATP-binding site and is involved in inter-ring interactions, and (3) an intermediate domain that connects the apical and equatorial domains (Braig et al. 1994). GroEL belongs to a class of macromolecules collectively termed "protein machines" since it undergoes nucleoside triphosphate binding and hydrolysis-driven ordered conformational changes (Ranson et al. 2001) that are crucial for its function. These conformational changes are responsible for its cycling between protein substrate-binding and -release states (Staniforth et al. 1994; Yifrach and Horovitz 2000). ATP-triggered conformational changes in GroEL are reflected in steady-state kinetic measurements of initial rates of its ATPase activity at different concentrations of ATP, which have shown that it undergoes two ATP-induced allosteric transitions: one with a midpoint at relatively low ATP concentrations and the second with a midpoint at higher ATP concentrations (Yifrach and Horovitz 1995). Each of the allosteric transitions reflects homotropic intraring positive cooperativity in ATP binding. The higher ATP concentration required to induce the second allosteric transition reflects homotropic inter-ring negative cooperativity in ATP binding. A nested allosteric model that describes these results has been proposed (Yifrach and Horovitz 1995) in which each ring of GroEL interconverts in a concerted (Monod et al. 1965) manner between a T state, with high affinity for nonfolded proteins and low affinity for ATP, and an R state, with low affinity for nonfolded proteins and high affinity for ATP. The intra-ring concerted transitions are nested in sequential (Koshland et al. 1966) ATP-promoted transitions of the GroEL double-ring from the TT state (both rings are in the T state) via the TR state to the RR state.

Protein (or peptide) substrate binding has been found to stimulate the ATPase activity of many different chaperones, including GroEL (Yifrach and Horovitz 1996), a member of the hsp60 family of molecular chaperones, and members of the hsp70 (e.g., BiP and hsc70; Flynn et al. 1989) and hsp90 (McLaughlin et al. 2002) families. The mechanisms responsible for the protein substrate-induced stimulation of the ATPase activity of molecular chaperones are, however, not known. Here, we show that, in the case of GroEL, this stimulation is due, at least in part, to modulation of its affinity for ADP by Glu257, a conserved (Brocchieri and Karlin 2000) residue at the N-cap of helix I in the apical domain. Mutational (Fenton et al. 1994) and crystallographic (Buckle et al. 1997; Chen and Sigler 1999) analyses have shown that helices H and I in the apical domain are involved in polypeptide substrate binding. In the apo state of wild-type GroEL (Bartolucci et al. 2005) and in one mini-chaperone (residues 191–336 of the apical domain) structure (Chen and Sigler 1999) but not in another (residues 191–376 of the apical domain; Buckle et al. 1997), Glu257 is involved in interactions with Asn229 and Ile230 in helix H. In the presence of a peptide substrate, the O{varepsilon}2 atom of Glu257 is found in one structure (Buckle et al. 1997) to be 2.78 Å away from the backbone NH of Gly-2 in the peptide with which it forms a hydrogen bond. In the other mini-chaperone–peptide complex structure (Chen and Sigler 1999), the O{varepsilon}1 atom of Glu257 is 2.52 and 3.12 Å away from the atoms C{zeta}2 of Trp2 and C{gamma} of Pro12 in the peptide, respectively. Residue Glu257 has also been implicated in interactions with protein substrates in a recent molecular dynamics simulation study (van der Vaart et al. 2004).


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Conclusions
 Materials and methods
 Appendix
 References
 
ATP hydrolysis in the presence of a nonfolded substrate
Given the apparent role of Glu257 in polypeptide substrate binding, we decided to test whether the mutation Glu257 -> Ala (E257A) affects stimulation of the ATPase activity of GroEL by nonfolded protein substrates. Initial rates of ATP hydrolysis by the Phe44 -> Trp (F44W) single mutant (a wild-type variant in which the F44W mutation was introduced so that ATP-induced conformational changes could be followed by monitoring time-resolved changes in fluorescence; Yifrach and Horovitz 1998) and the F44W, E257A double mutant were measured in the presence of calcium-depleted and reduced {alpha}-lactalbumin. This substrate was chosen since it remains nonfolded and soluble under the folding conditions of the ATPase assay (Yifrach and Horovitz 1996). It may be seen in Figure 1 that, in the presence of 500-fold excess nonfolded {alpha}-lactalbumin, the ATPase activity of the F44W mutant is stimulated by a factor of about 2.7, whereas the ATPase activity of the F44W, E257A double mutant is even slightly reduced. Gel-filtration experiments (Fig. 2) showed that the affinities of the F44W and F44W, E257A mutants for nonfolded {alpha}-lactalbumin are about the same (as similar amounts of nonfolded {alpha}-lactalbumin were bound to the two GroEL mutants under identical conditions), thus indicating that the absence of stimulation in the case of the F44W, E257A mutant is not due to reduced binding of this nonfolded substrate. GroES binding and its effect on the ATPase activity of GroEL were also found to be unaffected by the mutation (data not shown); however, the yield in assisted folding of rhodanese was found to be reduced relative to F44W GroEL by 65% (data not shown). Hence, we decided to examine the kinetic properties of the F44W, E257A mutant with respect to its ATPase activity in more detail.


Figure 1
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  		Figure 1. Stimulation of the ATPase activity of the F44W single mutant and the F44W, E257A double mutant of GroEL by nonfolded {alpha}-lactalbumin. The ATPase activities of the F44W single mutant (circles) and the F44W, E257A GroEL double mutant (squares), in the absence (open symbols) and presence (filled symbols) of nonfolded {alpha}-lactalbumin, were measured as described (Brune et al. 1994, 1998). Reactions were initiated by adding 100 µM ATP to a mixture of 10 nM GroEL oligomer, 12 µM phosphate-binding protein, and 5 µM nonfolded {alpha}-lactalbumin (when appropriate) in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 50 mM KCl, and 1 mM dithiothreitol. Experiments were carried out in triplicate at 25°C.

 


Figure 2
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  		Figure 2. Size-exclusion chromatography of free and GroEL(F44W)- or GroEL(F44W, E257A)-bound nonfolded {alpha}-lactalbumin. (A) Solutions (1.5 mL) of 10 µM nonfolded {alpha}-lactalbumin and 1 µM of the F44W or F44W, E257A GroEL mutants or 10 µM of nonfolded {alpha}-lactalbumin by itself in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 50 mM KCl, and 1 mM dithiothreitol were incubated for 5 min at room temperature and then separated on a Hiload 16/60 Superdex 75 column (Pharmacia) equilibrated in the same buffer. (B) SDS-PAGE analysis of samples corresponding to the five peaks in the elution profiles.

 
Steady-state ATPase activity at different concentrations of ATP
Initial rates of ATP hydrolysis by the F44W, E257A mutant were measured as a function of ATP concentration (Fig. 3A). The data were fitted to a Hill-type equation for two sequential allosteric transitions (Kafri et al. 2001). The values of the Hill coefficients in the case of this mutant were found to be 3.1 (±0.6) and 3.6 (±0.6) for the transitions at low and high ATP concentrations, respectively (the relatively large errors in the values of the Hill coefficient are due to the narrow range of concentrations at which the first plateau is observed). These values are similar within error to those of the F44W mutant that were found to be 2.6 (±0.3) and 3.3 (±1.0) for the transitions at low and high ATP concentrations, respectively. Surprisingly, however, the F44W, E257A mutant does not display the decrease in ATPase activity at high ATP concentrations that is observed in the case of wild-type GroEL and is another reflection of the inter-ring interaction (Fig. 3B). Such a decrease was also not observed in the case of single-ring GroEL (Inobe et al. 2001), the Arg13 -> Gly, Ala126 -> Val GroEL mutant with diminished inter-ring cooperativity (Aharoni and Horovitz 1996) or the eukaryotic chaperonin containing TCP-1 (CCT) (Kafri et al. 2001). The data in Figure 3A were, therefore, also fitted to an equation based on the nested model (Yifrach and Horovitz 1995; data not shown) in order to determine whether inter-ring negative cooperativity is disrupted in the F44W, E257A mutant. The values of L1 ([TR]/[TT]) and L2 ([RR]/[TR]) were found to be 0.015 (±0.005) and 5.6 (±5.0) x 10–8, respectively, indicating that this mutant has relatively intact inter-ring cooperativity with respect to ATP (L1/L2 = 2.7 (±2.6) x 105). It has been suggested that ADP binding to one ring inhibits ATP hydrolysis in the other ring (Kad et al. 1998), thereby explaining, perhaps, the decrease in ATPase activity at high ATP concentrations that is observed in the case of wild-type GroEL. We, therefore, decided to determine how ADP affects the ATPase activity of the F44W, E257A mutant.


Figure 3
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  		Figure 3. Steady-state ATPase activity of the F44W single mutant and F44W, E257A double mutant of GroEL as a function of ATP concentration. Initial velocities of ATP hydrolysis by the F44W, E257A (A) and F44W (B) mutants were measured as described (Horovitz et al. 1993) at different concentrations of ATP using a final GroEL oligomer concentration of 25 nM. The reactions were carried out in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 10 mM KCl, and 1 mM dithiothreitol at 25°C. The data for the F44W single mutant and the F44W, E257A double mutant were fitted to a Hill-type equation for two allosteric transitions (Kafri et al. 2001). The activity of the F44W mutant is higher than previously reported (Yifrach and Horovitz 1998) owing to the acetone purification step (Voziyan and Fisher 2000).

 
Transient kinetic analysis of ATP hydrolysis in the presence of ADP
Equal volumes of ATP and the F44W single mutant or the F44W, E257A double mutant were rapidly mixed using a stopped-flow device, and ATP hydrolysis was then followed as described (Brune et al. 1994, 1998). It may be seen in Figure 4A that a linear phase of ATP hydrolysis is preceded by a burst phase that is much more pronounced in the case of the F44W mutant (cyan trace) than the F44W, E257A mutant (green trace). The data are found to fit well to Equation A7 in the Appendix. The fits show that the amplitude of the burst phase in the case of the F44W mutant is –0.0112 (±0.0002) and, thus, 14-fold larger than the respective amplitude in the case of the F44W, E257A mutant, which is –0.0008 (±0.0001). In contrast, the values of the apparent rate constants corresponding to the burst phases of the F44W and F44W, E257A mutants are similar and found to be 0.54 (±0.02) and 0.86 (±0.33) sec–1, respectively. The values of the apparent rate constants corresponding to the linear phases of the F44W and F44W, E257A mutants are found to be about 0.012 and 0.014 (in arbitrary units per second), respectively, in agreement with the relative steady-state ATPase rates at 100 µM ATP determined with the radioactive assay (Fig. 3). In the model on which Equation A7 is based, the burst phase reflects the first round of ATP hydrolysis, which, in contrast to subsequent rounds, does not require displacement of ADP by ATP. Our results suggest, therefore, that the less pronounced burst phase in the case of the F44W, E257A mutant may be due to a more rapid off-rate of ADP. This interpretation is supported by the observation that the amplitude of the burst phase decreases by a factor of about 6 to –0.0018 (±0.0002) when the F44W mutant is preincubated with a final concentration of 200 µM ADP before mixing with ATP (Fig. 4, red trace). The value of the apparent rate constant corresponding to the linear phase of hydrolysis was also found to decrease by a factor of about four to 0.0029 (in arbitrary units per second), in the presence of 200 µM ADP, whereas the value of the apparent rate constant corresponding to the burst phase was found to be ~0.5 sec–1 and, thus, unchanged. In the case of the F44W, E257A mutant, in the presence of 200 µM ADP (purple trace), no burst phase is observed and the value of the apparent rate constant corresponding to the linear phase of hydrolysis is found to decrease by a factor of about two to 0.0086 (in arbitrary units per second). The values of the apparent rate constant corresponding to the linear phase and the amplitude of the burst phase were found to decrease monotonically with increasing ADP concentration (data not shown).


Figure 4
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  		Figure 4. Time courses of ATP hydrolysis by the F44W single mutant and F44W, E257A double mutant of GroEL. (A) The data from 0.125 (indicated by the broken vertical line) to 14 sec (data after 7 sec not shown) for the F44W single mutant in the absence (cyan) and presence (red) of 200 µM ADP and for the F44W, E257A double mutant in the absence of ADP (green) were fitted to Equation A7 in the Appendix and those for the F44W, E257A double mutant, in the presence of 200 µM ADP (purple), to a straight line (these fits are shown as continuous lines). The linear portions of the traces were also fitted to a straight line (dashed line) in order to highlight the burst phase when present. Plots of residuals with random deviations about zero indicate good fits of the data for the F44W, E257A (B) and F44W (C) mutants, in the absence of ADP, and for the F44W, E257A (D) and F44W (E) mutants, in the presence of ADP, to the above-mentioned equations. The fast transients before 0.125 sec were not analyzed, as they reflect contaminating phosphate that was not removed by the mop and is, thus, present before the start of the reaction. The red, purple, cyan, and green average traces were shifted vertically relative to each other and all have the same time zero. ATP hydrolysis was followed as described at a fixed final concentration of 100 µM ATP (Brune et al. 1994, 1998).

 
Steady-state ATPase activity at different concentrations of ADP
Next, we examined the steady-state ATPase activity of the F44W and F44W, E257A mutants at different concentrations of ADP and a fixed relatively low concentration of ATP so that only one ring undergoes the T -> R transition (Fig. 5). The data were fitted to Equation A10 in the Appendix, which takes into account the allosteric effects of ATP and ADP, competitive inhibition of ATP hydrolysis by ADP bound to the ring in the R conformation, and partial noncompetitive inhibition of ATP hydrolysis by ADP bound to the other ring, which is in an ADP-bound D state. The fits in Figure 5 yielded relatively large values for the allosteric constant L'1 ([DR]/[TR]) of 0.08 (±0.01) and 0.23 (±0.03) for the F44W and F44W, E257A mutants, respectively, that are consistent with the apparent noncooperative binding of ADP to GroEL (Inobe et al. 2001). The best fixed value of [S]/KR for the F44W mutant was found to be 4, in very good agreement with previously determined values of KR of about 10 µM (Yifrach and Horovitz 1995, 1998) and the 40 µM concentration of ATP that was employed. The value of [S]/KR for the F44W, E257A mutant was found to be 2.1 (±0.7) and, thus, also in very good agreement with the value of KR (that was determined from the data in Fig. 3A to be 14.9 (±1.2) µM) and the 40 µM concentration of ATP employed, from which a value of 2.7 (±0.2) is calculated for [S]/KR. It is clear from inspection of the data in Figure 5 that ADP inhibits the ATPase activity of the F44W mutant more efficiently than that of the F44W, E257A mutant. This is reflected in apparent 50% inhibition constants of about 49 and 225 µM for the F44W and F44W, E257A mutants, respectively. In the case of the F44W mutant, the values of KI and KI' (the respective ADP dissociation constants of the R and D states) are found to be 8.2 (±0.5) and 505 (±51) µM, respectively, in good agreement with the previously determined values of 5 (±2.4) (Burston et al. 1995) and ~700 µM (Kad et al. 1998) for these dissociation constants. In the case of the F44W, E257A mutant, the values of KI and KI' are found to be 33 (±3) and 806 (±150) µM, respectively, and, thus, higher than those of the F44W mutant. Surprisingly, the values of beta (kcat(DR)/kcat(TR)) for the F44W and F44W, E257A mutants are found to be 3.8 (±0.3) and 3.5 (±1.0), respectively, indicating that ADP binding to the ring in the D conformation in the DR state should have a stimulatory effect on the catalytic rate constant of this species. The values of the apparent catalytic rate constants (betaL1') for the F44W and F44W, E257A mutants are, however, 0.30 (±0.04) and 0.81 (±0.25), respectively, and, thus, consistent with the decrease in ATPase activity at high ATP concentrations that is observed in the case of the F44W mutant but not the F44W, E257A mutant.


Figure 5
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  		Figure 5. Inhibition by ADP of the steady-state ATPase activity of the F44W single mutant and F44W, E257A double mutant of GroEL. The ATPase activity of the F44W (open circles) and F44W, E257A (filled circles) GroEL mutants was measured as described (Brune et al. 1994, 1998) in the presence of a fixed concentration of 40 µM ATP and different concentrations of ADP in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 10 mM KCl, and 1 mM dithiothreitol at 25°C. The oligomer concentration of GroEL was 25 nM. Each data point is the average of three to six independent measurements. The data were fitted to Equation A10 in the Appendix using fixed values of L1 of 0.002 and 0.015 for the F44W and F44W, E257A mutants, respectively, that were determined by fitting the steady-state ATPase data in Figure 3 to an equation based on the nested model as described (Yifrach and Horovitz 1995).

 

   Conclusions
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
Materials and methods
 Appendix
 References
 
It is shown here that nonfolded protein substrate binding-induced stimulation of the ATPase activity of GroEL is abolished by the mutation E257A. Insight into the mechanism of such stimulation, which is common to many chaperone families, can, therefore, be gained by analyzing the properties of the E257A mutant. Taken together, the transient and steady-state kinetic data reported in this article indicate that different conformational states of the F44W, E257A mutant have a lower affinity for ADP as compared with the F44W wild-type variant. The transient kinetic data suggests that this lower affinity is due, at least in part, to a more rapid off-rate. Our data suggest, therefore, that Glu257-mediated nonfolded protein substrate binding to GroEL stimulates its ATPase activity by increasing the off-rate of ADP, thereby allowing more rapid turnovers of ATP binding and hydrolysis. How Glu257 in the apical domain communicates with the ~40 Å-removed nucleotide-binding site in the equatorial domain remains, however, unclear. An increase in the off-rate of ADP upon nonfolded protein substrate binding may also help explain how nonfolded protein substrate binding to one (the trans) ring accelerates the departure of GroES from the opposite (cis) ring, thereby speeding up the GroE reaction cycle (Rye et al. 1999). The absence of this effect in the case of the F44W, E257A mutant may explain why it is less efficient in assisting rhodanese folding. ADP release was also found to be rate limiting in the function of other molecular motors such as myosin (Weiss et al. 2001) and may, thus, be a common property of many ATP-fueled biomolecular machines.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
 Materials and methods
Appendix
 References
 
Mutant construction and protein purification
The GroEL F44W, E257A double mutant was generated as before (Horovitz et al. 1993) using single-stranded DNA of the plasmid pOA (Horovitz et al. 1993) containing the gene for the F44W GroEL mutant (Yifrach and Horovitz 1998) and the mutagenic oligonucleotide (E257A): 5'-AGTTGCCAGCGCTGCGCCTTCTACATC-3'. Expression of GroEL was carried out as described previously (Horovitz et al. 1993), and its purification was achieved as described (Yifrach and Horovitz 1998) followed by precipitation in 45% (v/v) acetone (Voziyan and Fisher 2000). Human {alpha}-lactalbumin was purchased from Sigma and further purified by gel filtration on a Hiload 16/60 Superdex 75 column (Pharmacia) equilibrated in 20 mM Tris-HCl buffer (pH 7.2) containing 100 mM NaCl. Purified {alpha}-lactalbumin was denatured as described previously (Yifrach and Horovitz 1996).

ATPase assays
Initial (steady-state) rates of ATP hydrolysis were measured using a radioactive assay (Horovitz et al. 1993) or as described (Brune et al. 1994, 1998) by monitoring time-resolved changes in fluorescence of coumarin-labeled phosphate-binding protein (PBP) at wavelengths longer than 455 nm (using a cutoff filter) upon excitation at 430 nm using an ISS PC1 spectrofluorimeter. Nucleotide solutions used in experiments with PBP were treated with a phosphate mop (500 µM MEG, 1 unit PNPase) as described (Brune et al. 1994, 1998) and the PNPase was then removed by filtering the nucleotide solution on a Centricon YM-50 concentrator. The method of Webb and coworkers (Brune et al. 1994, 1998) was also used for transient kinetic measurements of ATPase activity. These experiments were carried out using an Applied Photophysics SX.17MV stopped-flow apparatus with a 0.2 cm pathlength and entrance and exit monochromator wavelength bandpasses set to 7 nm. Reactions were initiated by mixing equal volumes of 50 nM GroEL oligomer (with 16 µM phosphate-binding protein and, when appropriate, 400 µM ADP) and 200 µM ATP in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 10 mM KCl, and 1 mM dithiothreitol at 25°C. Between five and 10 traces were collected for 50.5 sec (using a split time base of 0.5 and 50 sec with 2000 sampling points in each time interval) for each experimental condition and then averaged.


   Appendix
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
 Materials and methods
 Appendix
References
 
The analysis of the time course of ATP hydrolysis by GroEL is based on the scheme in Figure 6 that is further simplified, as follows:


Formula A1

where S and I stand for ATP and ADP, respectively, D stands for the ADP bound state of a GroEL ring, and T and R are defined as before. If ADP is incubated with GroEL prior to addition of ATP, then the following scheme applies:


Formula A2

where K = [TT][I]n/[TDIn]. Assuming that the TTSn species is in steady-state (i.e., [TTSn] = (k1/k2)[TT]) and from conservation of mass one has


Formula A3

where [E]T = [TT] + [TTSn] + [TRSn] + [TDIn]. Given Scheme A2 and Equation A3 one may write


Formula A4

where Formula and Formula .


Figure 6
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  		Figure 6. Scheme showing different allosteric states of GroEL. ATP binding (1) to the T state (that has high affinity for nonfolded substrates and low affinity for ATP) triggers a switch to the R state (2) with high affinity for ATP (conformational selection can also facilitate the T to R transition). ATP hydrolysis follows and is accompanied by an R to D conformational transition (3). Dissociation of ATP that is accompanied by a D to T conformational transition must occur before a new round of ATP hydrolysis can take place (4). In this scheme, ATP binding and hydrolysis by only one ring is considered.

 
Integration of Equation A4 yields


Formula A5

The rate of inorganic phosphate, Pi, release is given by


Formula A6

Integration of Equation A6 yields


Formula A7

In Equation A7 that is used to fit the traces in Figure 4, Formula and beta correspond to the steady-state and transient rates of hydrolysis, respectively. A result with the same form as Equation A7 is obtained for the scheme Formula (i.e., hydrolysis is due to the T state, which has lower affinity for ATP but a higher catalytic rate constant) without assuming that the TTSn species is in steady-state.

The analysis of the inhibition of the steady-state ATPase activity of GroEL by ADP (I) is based on the following binding polynomial (P):


Formula A8

where DR stands for a GroEL state in which one ring is in the R conformation and the other ring in an ADP-bound conformation designated D, L'1 ([DR]/[TR]) is the allosteric equilibrium constant for the transition TR -> DR, KR is the ATP dissociation constant of the R state, KI and KI' are the respective ADP dissociation constants of the R and D states, and all other symbols are defined as before. In this equation, the terms L1[TT](1 + [S]/KR + [I]/KI)7 and L'1L1[TT](1 + [S]/KR + [I]/KI)7 (1 + [I]/K'I)7 stand, respectively, for all the species in the TR state with ADP and ATP bound to the ring in the R conformation and all the species in the DR state with ADP bound to the ring in the D conformation and ATP and ADP bound to the ring in the R conformation. For simplicity, it is assumed in Equation A8 that ATP binds exclusively to the R state and that the transition TR -> RR can be neglected owing to the relatively low ATP concentration employed. The fractional saturation binding curve, Y, can be derived from the binding polynomial using the relationship


Formula A9

where N (7) is the total number of ATP binding sites. The initial ATPase velocity, V0, divided by the maximal initial ATPase velocity, Vm, can, therefore, be expressed, as follows:


Formula A10

where beta = kcat(DR)/kcat(TR) and Vmax = kcat(TR)P.


   Footnotes
 
1 These authors contributed equally to this work. Back

Reprint requests to: Amnon Horovitz, Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; e-mail: Amnon.Horovitz{at}weizmann.ac.il; fax: ++972-8-9344188.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062100606.


   Acknowledgments
 
This work was supported by The Israel Science Foundation. A.H. is an incumbent of the Carl and Dorothy Bennett Professorial Chair in Biochemistry. We thank Dr. M.R. Webb (National Institute for Medical Research, Mill Hill, London) for cells expressing A197C Escherichia coli phosphate-binding protein.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
 Materials and methods
 Appendix
 References
 
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Brune M., Hunter J.L., Corrie J.E.T., Webb M.R. 1994. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment I ATPase. Biochemistry 33: 8262–8271.[CrossRef][Medline]

Brune M., Hunter J.L., Howell S.A., Martin S.R., Hazlett T.L., Corrie J.E.T., Webb M.R. 1998. Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37: 10370–10380.[CrossRef][Medline]

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