Allosteric Enhancement of Adaptational Demethylation by a Carboxyl-terminal Sequence on Chemoreceptors*

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme.

Sensory systems are designed to adapt to persistent stimulation. In bacterial chemotaxis, adaptation is accomplished through covalent modification of transmembrane chemoreceptors (1). Specific glutamyl residues in the chemoreceptor cytoplasmic domain (2) are methylated by methyltransferase CheR (3) to form carboxyl methyl esters. These esters can be hydrolyzed by methylesterase CheB (4). In the well studied chemosensory systems of Escherichia coli and Salmonella enterica serovar typhimurium (see Refs. 5-7 for reviews), chemoreceptors have four to six methyl-accepting glutamyl residues, four of which are at conserved positions (2, 8 -11) (Fig. 1). Control of receptor methylation and demethylation determines steady state cellular behavior, the ability of the chemosensory system to provide exact adaptation over a wide dynamic range, and the process of molecular memory. Thus the means by which receptor modification is modulated are of great interest.
Chemoreceptors form complexes with the chemotaxisspecific histidine kinase CheA, enhancing an otherwise low rate of autophosphorylation (12) and providing phosphoryl groups for transfer to two response regulators, CheY and CheB (13). Phospho-CheY binds the flagellar rotary motor and determines the direction of rotation (14). CheB is phosphorylated on its regulatory domain, activating its catalytic domain (15) (Fig. 1). Binding of chemoattractant to receptor reduces kinase activity (12) and thus cellular levels of phospho-CheY, resulting in a change in rotational bias and thus an altered pattern of swimming (16). However, these changes are short-lived because an increase in ligand occupancy also initiates the feedback loop of adaptation. Stimulated receptors accumulate methyl groups through a combination of a reduced cellular level of phospho-CheB and occupancy specific activation of methyl-accepting sites (17,18). Increased methylation creates a compensatory change in the receptorkinase complex (19) that restores CheA activity to its null, receptor-activated state even though the increased level of attractant persists.
What determines the efficiency of adaptational methylation and demethylation? Sites of modification are spaced seven apart in the receptor sequence (2), are on solvent-exposed surfaces of the chemoreceptor cytoplasmic domain (20), and are bracketed by sequences that share common features and influence kinetic preferences among sites (8 -10, 21). However, a crucial determinant is at the chemoreceptor carboxyl terminus, separated from the sites of modification. In E. coli and Salmonella, a conserved sequence, asparagine-tryptophanglutamate-threonine-phenylalanine (NWETF), at the carboxyl terminus of high abundance chemoreceptors ( Fig. 1) interacts with the methyltransferase (22) and the methylesterase (23,24). Chemoreceptors lacking the pentapeptide naturally (the low-abundance receptors) or as the result of engineered truncations or mutations are inefficiently methylated, demethylated, and deamidated (23,(25)(26)(27)(28) and are ineffective on their own at mediating tactic response and directed movement (28 -32). Such receptors mediate effective taxis only with the assistance of NWETF-containing receptors (28,29,31,32).
How does the NWETF pentapeptide at the carboxyl terminus of a receptor enhance adaptational modification? For methyltransferase CheR, it provides a high affinity docking site that increases enzyme concentration near the methyl-accepting glutamates (22). In the current work we investigated enhancement of CheB-catalyzed demethylation by the pentapeptide sequence. We found that it occurs through a distinctly different mechanism of allosteric activation.

EXPERIMENTAL PROCEDURES
Proteins and Peptides-Procedures described previously were used to produce, purify, and quantify CheA, CheW, and membrane-embedded chemoreceptors (23,33), produce and purify CheB and CheBc (24), and synthesize and purify peptides (23). CheB was quantified using ⑀ 280 ϭ 13,700 M Ϫ1 cm Ϫ1 . This value was determined by quantitative amino acid analysis of Ͼ99% pure E. coli CheB obtained using a NWETF affinity column, with a S.E. Ϯ 1200. It was approximately midway between two rather different values for CheB published previously, one determined experimentally (34), the other calculated from amino acid composition (35). Because of these differences, we did five independent determinations by quantitative amino acid analysis, each using at least 10 amino acids, and obtained consistent values with a standard error Ͻ9%. As a control, we determined ⑀ 280 for bovine serum albumin (Pierce Chemical) and found a value within 10% of the accepted one. Concentrations of NWETF and related peptides were determined using the tryptophan ⑀ 279.8 ϭ 5,600 (36).
Receptor Demethylation-Kinetic analyses of demethylation were performed on forms of Tar with the gene-designated two glutamates and two glutamines ("2E-2Q") at the sites of adaptational modification, produced in cells lacking other chemotaxis proteins, isolated in native membrane, and methylated in vitro to 0.4 to 0.6 3 H-methyl groups per receptor monomer (23). In assay mixtures, phospho-CheB was generated by the presence of previously autophosphorylated CheA or by providing activated CheA in complex with chemoreceptor and CheW (23). Phosphorylation of CheB by receptor-activated CheA was quite effective (see "Results"); autophosphorylated CheA was somewhat less effective, as previously illustrated (23). To form activating complexes, 10 M membrane-embedded Tar⌬pp (2E-2Q), 5 M CheA, and 5 M CheW in 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM dithiothreitol, and 10% glycerol (TEDG) containing 50 mM KCl and 10 mM MgCl 2 (TEDGKM) were incubated at room temperature. After 1 h membrane vesicles containing 3 H-methyl-labeled receptors (Tar or Tar⌬pp) were added to final receptor concentrations from 1 to 10 M. 10 s later ATP and CheB were added to 1 mM and 0.05 to 0.5 M, respectively. At least three samples were taken over periods during which the rate of demethylation was essentially linear (60 to 180 s, depending on the particular experimental conditions). Initial rates were determined by linear least squares fits through the points. In experiments testing activation of Tar⌬pp demethylation by free peptides, receptor-CheA complexes were formed as above except that 10 M Tar⌬pp used to form complexes with CheA and CheW carried 3 H-methyl groups that provided the substrate for phospho-CheB, no additional methylated receptors were added to the mixture after the 1-h incubation, and a peptide was present from the beginning of the incubation at concentrations ranging from 0.4 to 1.6 mM.
Binding of Isolated NWETF to CheB-Isothermal titration calorimetry was performed with a MicroCal Omega instrument (MicroCal Inc., Northampton, MA). The data were analyzed using a single-set-of-sites model from which were obtained values for binding stoichiometry and association constants. Equilibrium dialysis was performed using radiolabeled pentapeptide NWETF and pure CheB. Additions of amino acids to the pentapeptide amino terminus do not have a detectable effect on interaction with CheB (23), so we introduced radiolabel by modification of the amino-terminal amino group with N-succinimidyl [2,3-3 H]propionate (Amersham Biosciences). Radiolabeled reagent at ϳ1 mCi/ml was added to pure NWETF peptide at 10 mg/ml in 50 mM sodium phosphate buffer, pH 7.2. After incubation overnight, the mixture was dialyzed extensively against TEDGKM. Radiolabeled pentapeptide was mixed with nonradioactive pentapeptide to create specific activity ϳ3 cpm/ pmol. For binding assays, 100-l samples of an ϳ50 M solution of CheB in TEDGKM were placed in dialysis tubing (Spectrapore, molecular weight cutoff ϭ 14,000), sealed at one end by a Spectrapore closure and the other end stretched over a piece of plastic tubing. The bag was immersed in 5 ml of a stirred solution of radiolabeled peptide in TEDGKM at 4°C. Dialysis was continued until equilibrium was reached as judged by samples taken in the course of the dialysis from the open end of the tube (ϳ2 days). At equilibrium, the amount of pentapeptide bound to CheB was determined from duplicate determinations of the difference between radioactivity inside the dialysis bag and in the dialysis buffer.
Binding of Receptor-borne NWETF to CheB-Studies were performed using radiolabeled CheB-S164C produced in strain RP3098 harboring a form of pCW/cheB (24) in which codon 164 was altered to code for cysteine, thus eliminating catalytic activity (37). Cells were grown in a minimal salts medium to ϳ3 ϫ 10 8 cells/ml and isopropyl-1-thio-␤-Dgalactopyranoside was added to 0.25 mM. One hour later, a L-[U- 14 C]amino acid mixture (Amersham Biosciences) was added. After 10 min the culture was rapidly cooled in an ice-water bath, the cells were processed and CheB was purified as described (23,33). Membrane vesicles containing Tsr or Tar were produced as described (33). For experiments using vesicles with a high content of Tsr, membranes were purified on a sucrose gradient (35). Binding assays were based on those of Gegner et al. (35). 50-l mixtures of receptor-containing membranes, ϳ25 M radiolabeled CheB-S164C, and 0.5 mg/ml bovine serum albumin in TEDG were incubated 10 min at room temperature and centrifuged at 50,000 rpm for 15 min in a TLA100.4 rotor. Two 20-l samples of the resulting supernatant were analyzed by liquid scintillation counting. In these experiments, receptor was not methylated and thus there were no methyl ester substrate sites to which CheB might bind.

RESULTS
Kinetic Measurements-Previously, we compared demethylation of Tar and Tar⌬pp, its truncated derivative lacking the NWETF sequence, at a single receptor concentration and found that rates of CheB-catalyzed demethylation for intact receptor were at least 20-fold greater than rates for receptor lacking the pentapeptide sequence (23). This was the case whether CheB was in the absence or presence of a means of generating its activated form, phospho-CheB (23). We were interested in performing more extensive kinetic analyses comparing demethylation of Tar and Tar⌬pp by determining initial velocities of demethylation over a range of substrate concentration. A quantitative comparison was not possible for demethylation catalyzed by unphosphorylated CheB because demethylation of Tar⌬pp was so ineffective that reliable rates could not be obtained to compare with the substantially faster rates of Tar demethylation (see Ref. 23 for an example).
However, quantitative comparison could be made for demethylation reactions catalyzed by phospho-CheB. Plots of initial velocity as a function of substrate (methyl group) concentration (Fig. 2) document the impressive enhancement of receptor demethylation provided by the carboxyl-terminal sequence. A double reciprocal plot (inset Fig. 2) illustrates that this enhancement is the result of a difference in V max (the y axis intercept) and not in K m (x axis intercept). These differences were seen whether phospho-CheB was produced using phosphorylated CheA or by providing CheA activated in complex with chemoreceptor (see "Experimental Procedures"). The latter protocol was more efficient in activation (23), but in both protocols, phospho-CheB was created independently of the methylated receptor substrates that were being compared. We repeated experiments like those in Fig. 2, testing several concentrations of CheB. From three such experiments, we calculated the mean values (Ϯ S.E.) for K m and k cat , which is V max normalized to the amount of enzyme. For Tar, K m ϭ 2.75 Ϯ 0.9 M and k cat ϭ 187 Ϯ 49 mmol of methanol s Ϫ1 mol CheB Ϫ1 ; for Tar⌬pp, K m ϭ 2.3 Ϯ 0.5 M and k cat ϭ 7.9 Ϯ 1.7 mmol methanol s Ϫ1 mol CheB Ϫ1 . Thus the presence of receptor-borne NWETF increased catalytic efficiency, as measured by k cat , over 20-fold but did not alter the apparent affinity of enzyme for substrate, as reflected in K m . Because phospho-CheB is short lived, only a fraction of the CheB in our experiments would have been in the phosphorylated form (38), and thus the k cat values we determined are underestimates of the true values (39). However, because the level of phospho-CheB would have been the same whether the methylated substrate was Tar or Tar⌬pp, the difference between the k cat values we determined for the two forms of the receptor provided a valid indication of the effect of the receptor carboxyl-terminal pentapeptide. In addition, the k cat value we determined for demethylation of Tar (ϳ190 mmol of methanol s Ϫ1 mol of CheB Ϫ1 ) is comparable with the value for Tar determined by Anand and Stock (ϳ150 mmol of methanol s Ϫ1 mol CheB Ϫ1 ) using phosphoramidate to create a steady state of ϳ65% phosphorylated CheB (39). This implies that our conditions generated a similar fraction of phospho-CheB.
If interaction of CheB and the receptor-borne sequence enhanced demethylation by changing k cat but not K m , then the enzyme missing the pentapeptide-binding site should catalyze demethylation of Tar and Tar⌬pp at essentially the same velocities and with a K m essentially unchanged from the intact enzyme. We tested this using CheBc, the carboxyl-terminal catalytic domain of the enzyme (Fig. 1) that is missing the regulatory domain and linker and thus lacks the pentapeptidebinding site at the juncture of those domains (24). As shown in Fig. 3, CheBc catalyzed demethylation of Tar and Tar⌬pp with almost the same efficiency. This was a striking contrast to the vast difference in velocities for the two substrates when demethylated by intact enzyme (Fig. 2). In addition, the K m values for CheBc catalysis as determined in multiple experiments (2.1 Ϯ 0.7 and 2.5 Ϯ 0.5, for Tar and Tar⌬pp, respectively) were essentially unchanged from the values for intact phospho-CheB (2.75 Ϯ 0.9 and 2.3 Ϯ 0.5 M, respectively) or for the value for intact but unphosphorylated CheB acting on Tar (2.9 Ϯ 1.4 M).
Binding of CheB to Pentapeptide NWETF-Wu et al. (22) used isothermal titration calorimetry to determine that CheR bound to the isolated NWETF pentapeptide in a 1:1 complex with a K d ϳ 2 M. We used the same procedure to characterize binding of CheB to the identical peptide and found a stoichiometry close to 1:1 (1:0.85) but a K d of ϳ160 M, indicating much weaker binding (Fig. 4). This weak interaction was unexpected so we investigated binding by a second technique, equilibrium dialysis. We labeled NWETF by amino-terminal modification with [ 3 H]propionate and used the radiolabeled pentapeptide in equilibrium dialysis experiments with CheB (Fig. 5). Analysis of the binding revealed a stoichiometry of 1:1 and a K d of 130 M, confirming that CheB bound the receptor pentapeptide much less strongly than did CheR.
We considered the possibility that pentapeptide sequence at its natural location, the carboxyl terminus of a chemoreceptor, might be bound more strongly by CheB than free peptide. Thus we tested binding of radiolabeled CheB to the NWETF-bearing chemoreceptor Tar, assaying depletion of radiolabel from a binding mixture after centrifugation of receptor-containing vesicles (35). If CheB bound to receptor-borne pentapeptide with an affinity comparable with the 2 M K d of the CheRpentapeptide complex, then assays with receptor-borne pentapeptide at 1-10 M should have revealed substantial binding. This was not the case, so we investigated higher concentrations using Tsr-containing membranes, which can be isolated with higher receptor content (35). Binding was detected for Tsrborne pentapeptide at concentrations above 10 M, but there were difficulties in testing pentapeptide concentrations Ͼ50 M because the volume of membrane vesicles became technically limiting. At 50 M receptor-borne pentapeptide, binding was not close to saturation. Extrapolation from the limited data provided estimates of K d ϳ 150 M and stoichiometry ϳ1:1, consistent with values for free pentapeptide.
Allostery-Studies of both binding and kinetics indicated that interaction of CheB and receptor-borne pentapeptide sequence enhanced demethylation by affecting catalysis, not by increasing local enzyme concentration. The pentapeptideenzyme interaction occurs at a site separated from the catalytic interaction, so this is an allosteric effect. The substrate as well as the enzyme is a protein, thus allostery could alter CheB, the protein substrate or both.
If the principal effect of pentapeptide-CheB interaction were allosteric activation of the enzyme, then it should not be crucial whether the interacting sequence were attached to the receptor or present as a free peptide. In such a case, CheB would be activated for enhanced demethylation of Tar⌬pp by the presence of sufficient free pentapeptide to occupy its binding site on CheB. To test this possibility, we compared demethylation of Tar⌬pp in the absence and presence of NWETF at concentrations 5-10-fold above its K d , but observed little difference (data not shown). It was possible that NWETF binding did not induce an allosteric activation because the amino end of the sequence had a charged amino group not present in the receptor-borne sequence. Thus we tested DPNWETF, the NWETF sequence extended at its amino terminus by the two amino acids that occur naturally at those positions in native Tar and is effective in binding CheB (23). The extended peptide caused only marginal activation of Tar⌬pp demethylation. We then considered the possibility that introduction into an assay mixture of a charged peptide at concentrations up to 2 mM could inhibit demethylation nonspecifically, masking specific activation. For this reason, we utilized control peptides with inverted sequences (FTEWN and FTEWNPD) that should have the same nonspecific effects as the natural sequences but do not bind to CheB (23). At concentrations from 0.1 to 2 mM, these peptides reduced the rates of Tar⌬pp demethylation only slightly (ϳ20% at the highest concentrations). Thus even after adjusting for the possible nonspecific effect, the natural peptide sequences caused little activation. For example, as illustrated in Fig. 6 demethylation of Tar⌬pp by phospho-CheB was hardly affected by the presence of a potentially activating peptide (the extended pentapeptide DPNWETF) in comparison to activity in the presence of the control, inverted peptide. The peptide caused a ϳ0.3-fold enhancement (0.28 Ϯ 0.12 in six measurement using four different preparations of membrane-embedded receptor), less than 2% of the ϳ25-fold enhancement provided by receptor-borne pentapeptide (Fig. 6, right-hand bar).
The minimal effect of free peptide implied that allosteric enhancement of demethylation required attachment of the NWETF sequence to the receptor. If this were the case, then free pentapeptide should act as an inhibitor for demethylation of an NWETF-bearing receptor. We investigated inhibition using the NWETF pentapeptide, its seven-residue, extended form, and the corresponding inverted-sequence peptides to control for nonspecific effects. As seen in a representative experi- The bars show normalized initial rates of demethylation for methylated receptor in the absence of the conserved peptide sequence (control), in the presence of free peptide at concentrations sufficient for substantial binding to the enzyme (free), and with the peptide at its natural location at the carboxyl terminus (attached). In six experiments using four preparations of 3 H-methyllabeled Tar⌬pp, demethylation rates were determined in the presence of peptide DPNWETF, the extended form of pentapeptide NWETF (free), or FTEWNPD, which has an inverted sequence (control), and were expressed relative to the "control" rate. In three experiments the peptides were at 0.8 mM and in three they were at 0.95 mM. The first two bars show the control rate (normalized to 1 and thus without error bars) and the mean, normalized rate and standard deviation (1.28 Ϯ 0.12) in the presence of free peptide. The third bar displays the rate of demethylation catalyzed by phospho-CheB for Tar, a receptor carrying the NWETF sequence at its carboxyl terminus, relative to the rate for Tar⌬pp, a form of that same receptor lacking the sequence. This relative rate (23.7 Ϯ 6.2) was calculated from values in the text. ment (Fig. 7), the native sequence inhibited demethylation of intact Tar catalyzed by phospho-CheB. Inhibition was halfmaximal at a peptide concentration ϳ4-fold above the 150 M k d of the peptide-enzyme complex, as might be expected for competition with pentapeptide tethered to receptor and thus present at a high effective concentration (40). In any case, the high concentrations of free peptide necessary to inhibit demethylation by phospho-CheB (Fig. 7), indicated that phosphorylated enzyme did not have a substantially higher affinity for pentapeptide than the ϳ150 M k d we measured for unphosphorylated CheB (Figs. 2 and 3). DISCUSSION We found that enhancement of CheB-catalyzed demethylation by the NWETF pentapeptide sequence at the carboxyl terminus of a chemoreceptor occurred in a way distinctly different from its enhancement of CheR-catalyzed methylation. For the methylation reaction, the sequence provides a high affinity docking site for the CheR methyltransferase and thus increases the effective concentration of the enzyme in the neighborhood of substrate methyl-accepting sites (22). In contrast, interaction of CheB with receptor-borne pentapeptide enhanced demethylation allosterically. Consistent with an allosteric action, the sequence bound too weakly to the CheB methylesterase to cause a significant increase in local concentration of the enzyme.
Different Mechanisms of Enhancement-Of the two kinds of CheR-interaction sites on a chemoreceptor, the strongest binding is not at the sites of catalysis (the methyl-accepting glutamyl residues) but instead at the carboxyl-terminal NWETF sequence. CheR binds with the same dissociation constant of ϳ2 M to pentapeptide alone, to the isolated cytoplasmic domain of chemoreceptor Tsr carrying the pentapeptide sequence and three methyl-accepting sites, and to intact receptor carrying the sequence and from one to five methyl-accepting sites, but exhibits no detectable binding to isolated cytoplasmic domains containing methyl-accepting sites but missing the carboxyl-terminal sequence (22). Differences in rates of methylation for Tar and Tar⌬pp imply that binding of CheR to its substrate sites is ϳ20-fold weaker than binding to the pentapeptide sequence (26). Taken together, these observations show that the sequence acts as a high affinity docking site for the methyltransferase, enhancing the methylation reaction by recruiting the enzyme to the vicinity of its substrate sites and thus increasing the probability of binding at the methyl-accepting glutamyl residues.
Our data indicate that this is not the mechanism by which receptor-borne pentapeptide enhances CheB-catalyzed demethylation. Kinetic analysis of demethylation revealed that the NWETF sequence at the carboxyl terminus of a receptor substrate enhanced k cat not K m , and thus indicated that pentapeptide-enzyme interaction affected catalysis not binding. It is interesting to note that phosphorylation, which activates CheB independent of interaction with the pentapeptide (23), also enhances k cat rather than K m (39). Enhancement of k cat not K m was consistent with the weak binding we observed for the enzyme and pentapeptide sequence, a binding insufficient to enhance enzyme action by increasing concentrations of CheB near substrate sites. As documented in Figs. 4 and 5, CheB bound to the free pentapeptide NWETF with a K d ϳ 150 M, and the concentrations of free peptide necessary to inhibit phospho-CheB activity (Fig. 7) indicated that phosphorylation of the enzyme did not make the affinity substantially stronger. Investigation of CheB binding to the sequence at its natural location at the carboxyl terminus of a chemoreceptor indicated a similarly weak interaction. The short lifetime of phospho-CheB kept us from performing direct measurements of binding of phosphorylated enzyme to receptor-borne pentapeptide, but a strong binding for only this combination could not explain enhancement by the receptor-borne pentapeptide of demethylation catalyzed by CheB that is not phosphorylated (23), nor the effect of the receptor-borne pentapeptide on k cat rather than K m . Estimates of the cellular content of chemoreceptors range from 2,500 to 10,000 (41)(42)(43) and the content of CheB has been estimated at 2,000 (44). These values translate to cellular concentrations between 4 and 16 M for chemoreceptors and ϳ3 M CheB. At such concentrations, a CheB-pentapeptide complex with a K d of ϳ150 M, would involve only a small proportion of total cellular CheB and this complex would not create a significant increase in concentration of CheB in the neighborhood of its sites of enzymatic action.
There is a disparity between the ϳ150 M K d of pentapeptide and CheB or phospho-CheB and the K m of ϳ3 M for demethylation by either CheB or phospho-CheB. We do not fully understand this disparity, but it can be understood if we assume that K m approximates K d for enzyme and substrate methyl ester (see below for the conditions under which this assumption would be valid). In this case, the interaction of the enzyme and substrate site would be significantly stronger that the interaction of the enzyme and receptor-borne pentapeptide. This stronger binding, as reflected in K m , would not be a function of the presence or absence of the pentapeptide sequence on a receptor (this is what we observe), and allosteric enhancement of CheB-mediated catalysis would be the result of binding of pentapeptide to enzyme bound to or near the substrate site. Using K m to approximate K d is valid if the rate constant of dissociation for unreacted substrate from the enzyme-substrate complex is substantially higher than k cat and the catalytic mechanism does not involve a covalent intermediate. These issues are currently under investigation. No matter what the affinity of enzyme for its substrate sites, the weak affinity of enzyme for pentapeptide indicates that this interaction could not assist CheB in passing through a receptor array by the "molecular brachiation" process postulated for CheR (40).
Kinetic parameters for phospho-CheB acting on NWETF- Reactions were initiated by addition of CheB and ATP to 0.08 M and 1 mM, respectively. For each concentration of extended peptide, initial rates of demethylation were expressed as a percentage of the rate in the presence of the control peptide and plotted as a function of peptide concentration. The curve is a fit of the data to a model that assumes that the initial rate is directly proportional to the fraction of CheB interacting with receptor-borne pentapeptide and that the extended peptide acts as a competitive inhibitor of that interaction.
containing receptor, K m ϳ 2.8 M and k cat ϳ 190 mmol of methanol s Ϫ1 mol of CheB Ϫ1 , are quite similar to the parameters for CheR, which are K m ϭ 2.1 M and k cat ϭ 170 mmol s Ϫ1 mol of CheR Ϫ1 (44). This is consistent with the dynamic equilibrium of adaptational modification, in which the population of unstimulated or adapted receptor is maintained at a constant level of methylation by a balance between rates of methylation and demethylation (45).
Allosteric Activation of Demethylation-The pentapeptidebinding site on CheB is separated from the active site (24) and thus enhancement of catalysis through an effect on the enzyme would be an effect of binding at a site distance from the site of catalysis, i.e. an allosteric effect. However, the substrate itself is a protein, so it was possible that interaction of its carboxylterminal sequence with the methylesterase could allosterically alter the receptor to enhance demethylation. The data are consistent with this second alternative, that allosteric activation is principally through the receptor, not the enzyme. If binding of the NWETF sequence induced an allosteric activation in the methylesterase, then it should not be crucial whether this sequence were present in its natural location at the carboxyl terminus of a chemoreceptor or as a free pentapeptide. Fig. 6 documents that free pentapeptide provided only a few percent of the activation created by the receptor-borne sequence. If allosteric activation required that the pentapeptide sequence be coupled to the chemoreceptor, then free pentapeptide should inhibit demethylation of receptors carrying the carboxyl-terminal sequence. Fig. 7 documents that this is the case. Taken together, these two results imply that the vast majority of the enhancement of demethylation generated by interaction of the receptor-borne sequence and CheB is conveyed through a change in the receptor, not the enzyme. Thus the primary allosteric effect appears to be through the protein substrate. However, the issues require additional investigation, because we have not yet demonstrated directly induction of a conformation change in the receptor by interaction of CheB and receptor-borne pentapeptide. Such investigations are underway.
Substrate-transmitted Allostery and Signal Amplification-Chemotactic response in E. coli exhibits striking signal amplification (46 -49). The mechanism of this amplification is not yet understood, but recent observations have implicated methylesterase CheB as a crucial participant (48,49). If CheB binding to receptor-borne, carboxyl-terminal pentapeptide induces substrate-transmitted allostery, this conformational change could be related to the role of CheB in signal amplification.