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J. Biol. Chem., Vol. 275, Issue 27, 20302-20307, July 7, 2000

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Thermal Repair of Tryptophan Synthase Mutations in a Regulatory Intersubunit Salt Bridge

EVIDENCE FROM ARRHENIUS PLOTS, ABSORPTION SPECTRA, AND PRIMARY KINETIC ISOTOPE EFFECTS^*

Ying-Xin Fan, Peter McPhie, and Edith Wilson Miles^§

From the Section on Enzyme Structure and Function, Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830

Received for publication, May 1, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase ₂₂ complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between  subunit Asp⁵⁶ and  subunit Lys¹⁶⁷ play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an  subunit ligand, -glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, "open" conformation to a high activity, "closed" conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that  subunit Asp⁵⁶ and  subunit Lys¹⁶⁷ are not essential for catalysis and for allosteric communication between the  and  subunits but that their mutual interaction is important in stabilization of the active, closed form of the ₂₂ complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

An enzyme must undergo conformational transitions to selectively stabilize each intermediate along a reaction pathway. Catalysis can be modulated by factors that change the enzyme conformation or alter the equilibrium distribution of preexisting conformations. These factors include ligands, solvents, chaotropic agents, temperature, and single site mutations. In the present work, we ask whether factors that stabilize the active conformation of an enzyme can also overcome the deleterious effects of mutations.

We have recently investigated the effects of temperature on the activity of the tryptophan synthase ₂₂ complex (EC 4.1.2.20) in the reaction shown in Scheme I (1). We found that the conditions that yield nonlinear Arrhenius plots of the activity data also yield temperature-dependent changes in the equilibrium distribution of the E-Ser and E-AA intermediates shown in Scheme I and in primary kinetic isotope effects. The results provide evidence for a temperature-induced conversion from a low activity open conformation to a high activity closed conformation (1). Different monovalent cations, which bind to the  subunit, and an allosteric ligand (DL--glycerol 3-phosphate (GP)),¹ which binds to the  subunit, affect the equilibrium distribution of the open and closed forms. Here we investigate the effects of increased temperature and of other factors that stabilize the active, closed conformation of tryptophan synthase on the effects of mutations in residues that can form a regulatory salt bridge between the  subunit and the  subunit.²

View larger version (9K): [in this window] [in a new window]   Scheme I.   Enzyme substrate intermediates in the reaction of the tryptophan synthase 22 complex with L-serine and indole. The reaction of the 22 complex with L-serine yields the pyridoxal 5'-phosphate aldimine of L-serine (E-Ser), which loses the -proton to form a quinonoid intermediate, E-Q1, followed by elimination of the hydroxyl group to yield the aldimine of aminoacrylate (E-AA). In the absence of indole, an equilibrium mixture of the E-Ser (max = 424 nm) and E-AA (max = 340 nm) intermediates accumulates (Stage I). In the presence of indole, E-AA reacts with indole to form a quinonoid intermediate (E-Q2), which is then protonated to form the aldimine of the product, L-tryptophan (E-Trp) (Stage II). The intermediates have characteristic spectroscopic properties (26-29). It has been proposed that allosteric signals derived from ligand binding or reactions at the two active sites switch the 22 complex from an open, low activity state to a closed, high activity state (8, 24, 30, 31).

Three-dimensional structures of wild-type and mutant forms of the tryptophan synthase ₂₂ complex from Salmonella typhimurium have revealed many features including the arrangement of the  and  subunits, the location of residues in the interface between the  and  subunits, and a monovalent cation binding site in the  subunit (2-7). Although Na⁺, K⁺, and Cs⁺ bind to the same site in the  subunit, differences in coordination give rise to two distinctly different protein conformations (3) (reviewed in Ref. 8, Scheme II). In the presence of Na⁺, the carboxylate of Asp³⁰⁵ forms a salt bridge with the -amino group of Lys¹⁶⁷ (Scheme IIA). In the presence of K⁺ or Cs⁺, the carboxylate of Asp³⁰⁵ is in an alternative conformation and the -amino group of Lys¹⁶⁷ moves about 7 Å and makes a salt bridge across the subunit interface with the carboxylate of Asp⁵⁶ (Scheme IIB). Previous studies have shown that the activities of enzymes having mutations at Lys¹⁶⁷ or Asp⁵⁶ are much higher in the presence of Cs⁺, K⁺, or NH₄⁺ than in the presence of Na⁺ (9, 10).³ These results suggest that certain ligands can repair the harmful effects of mutation by stabilizing the active conformation of the mutant enzymes.

View larger version (11K): [in this window] [in a new window]   Scheme II.   Alternative salt bridges between Lys167 and Asp305 (A) or between Lys167 and Asp56 (B). Salt bridge A is found in the wild type 22 complex in the presence of Na+ (3). Salt bridge B is found in the wild type 22 complex in the presence of Cs+ (3) or in the K87T 22 complex in the presence of L-serine and GP (4).

In the present work, we have compared the effects of temperature, monovalent cations (Na⁺ and Cs⁺), and an allosteric ligand (GP) on the wild type ₂₂ complex and on two mutant ₂₂ complexes, D56A and K167T. The results provide evidence that the mutagenesis of the residues involved in the salt bridge switch alters the catalytic and allosteric properties of the ₂₂ complex. Increased temperature in the presence of CsCl or of NaCl plus GP reversed the deleterious effects of the mutations. The results indicate that the  subunit Asp⁵⁶ and  subunit Lys¹⁶⁷ are not essential for catalysis and for allosteric communication between the  and  subunits but that their mutual interaction is important in stabilization of the closed form of the ₂₂ complex.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and Buffers-- L-Serine, DL-serine, GP, pyridoxal 5'-phosphate, and Bis-Tris propane were from Sigma. [-²H]DL-Serine was prepared as reported previously (11). Sodium-free GP was prepared by repeated passage of the solution of the disodium salt over an ion exchange column (DW-50 in the H⁺ form, Sigma) (12); the pH of the final eluate was adjusted to 7.8 with Bis-Tris propane. All experiments were carried out in 50 mM Bis-Tris propane buffer containing 0.5 mM dithiothreitol. The pH of this buffer was adjusted to 7.8 by the addition of HCl at 25 °C with no compensation for pH variation with temperature (1).

Bacterial Strain, Plasmids, and Enzymes-- Wild type and mutant plasmids pEBA-10 and pEBA-4A8 (13) were used to express wild type and D56A tryptophan synthase ₂₂ complex and the  subunit from S. typhimurium, respectively, in Escherichia coli CB 149 (14), which lacks the trp operon. The wild type and mutant ₂₂ were purified to homogeneity as described previously (15). The wild type  subunit was purified from extracts containing the  subunit alone (13, 16). The D56A  subunit was separated from the apo ₂₂ complex (17). The K167T ₂₂ complex was obtained as described previously (9). The enzymes were dialyzed against monovalent cation-free Bis-Tris propane buffer, pH 7.8, before use. Protein concentrations were determined from the specific absorbance at 278 nm using A_{1 cm}^1% = 6.0 for the ₂₂ complex and A_{1 cm}^1% = 4.4 for the  subunit (18).

Enzyme Assays and Spectroscopic Methods-- One unit of activity is the formation of 0.1 µmol of product in 20 min at the indicated temperature. The activity in the -replacement reaction with L-serine and indole was measured by a direct spectrophotometric assay (18) in the presence of excess  subunit (5 µM). [-¹H]DL-Serine and [-²H]DL-serine were used in the place of L-serine for investigations of primary kinetic isotope effects. The reaction mixtures were equilibrated for at least 5 min at the desired temperature before the addition of the enzymes. The concentration of L-serine (50 mM) used for assays was saturating because the same initial rates of the wild type or the mutant ₂₂ complexes were obtained with 25, 50, and 100 mM L-serine at 5 and 45 °C.

Absorption spectra and assays were measured using a Hewlett-Packard 8452 diode array spectrophotometer thermostated by a Peltier junction temperature controlled cuvette holder, which was calibrated with a thermometer. Buffers containing indicated components were equilibrated at the desired temperature for at least 5 min; spectra were obtained immediately upon the addition of a twentieth volume of the enzyme.

Data Analysis-- The activity data were plotted as logarithm of activity (ln Activity) versus the reciprocal of the absolute temperature in K (1/T) and fitted to the Arrhenius equation, Equation 1,

(Eq. 1)

where E_a is the energy of activation, R is the gas constant, and Z is the preexponential factor.

The results can be readily transformed into a rate constant, k_cat.⁴ According to Eyring's transition state theory, the temperature dependence of a rate constant is given by Equation 2,

(Eq. 2)

where k_B is the Boltzmann's constant, h is Planck's constant, and G, H, and S are the free energy, enthalpy, and entropy of activation of the rate-limiting step in the reaction, respectively. From Equations 1 and 2, the following can be shown (19).

(Eq. 3)

Thus, H and S can be determined from the Arrhenius plots.

The equilibrium constant, K_eq, for the E-Ser to E-AA conversion can be calculated from the temperature dependence of the absorbance using Equation 4,

(Eq. 4)

where A_T is the absorbance of the solution at absolute temperature T, A_E-AA and A_E-Ser are the absorbance of E-AA obtained at higher temperature and of E-Ser obtained at low temperature, respectively.

The temperature dependence of K_eq is given by Equation 5,

(Eq. 5)

where H_eq and S_eq are the changes in enthalpy and entropy of the reaction, respectively, and T_m is the midpoint, where K_eq(T) = 1. Assuming no change in enthalpy or entropy with temperature, we can obtain H_eq and S_eq from the linear plot of lnK_eq versus 1/T; T_m = H_eq/S_eq.

	RESULTS

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The D56A and K167T Mutations Alter the Temperature Dependence of the Activity of the ₂₂ Complex-- Fig. 1, AC, shows the effects of temperature on the activities of the wild type ₂₂ complex (1) and of the D56A and K167T ₂₂ complexes, respectively, in the presence of either NaCl or CsCl and in the absence or presence of the allosteric ligand, GP. Arrhenius plots of the data are shown in Fig. 1, D-F. At low temperatures (5-35 °C), the activities of the wild type ₂₂ complex were much higher in the presence of CsCl than in the presence of NaCl (1). However, at temperatures above 35 °C, the activities were similar in the presence of NaCl or CsCl (Fig. 1A). The Arrhenius plots (Fig. 1D) of the data were nonlinear in the presence of NaCl but were linear in the presence of CsCl, GP plus NaCl, or GP plus CsCl.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of temperature on the wild type and mutant 22 complexes in the presence of NaCl or CsCl and in the absence or presence of GP. The initial rates of the wild type (WT) (A), D56A (B), and K167T (C) 22 complexes in reaction with L-serine and indole were plotted as specific activity (units/mg) versus temperature in °C in the presence of 0.1 M NaCl or CsCl and in the absence or presence of 50 mM GP as indicated. The data from A, B, and C, respectively, were plotted as the logarithm of activity (ln Activity) versus the reciprocal of the absolute temperature in K (D, E, and F). The data were fitted to the Arrhenius equation (Equation 1). In cases where the plots were nonlinear, the linear portions at low or high temperatures were fitted to the Arrhenius equation separately. Activities were measured in the presence of excess wild type (A and C) or D56A (B)  subunit (0.5 µM). The concentration of the 22 complexes varied from 0.4 mg/ml (~5.5 µM pair) for the conditions giving lowest activity (e.g. either mutant enzyme in NaCl at low temperature) to 5 µg/ml (0.07 µM pair) for the conditions giving highest activity (e.g. all enzymes in CsCl at high temperature). The results for the wild type 22 complex are taken from Ref. 1.

The activities of the D56A and K167T ₂₂ complexes were very low in the presence of NaCl but were dramatically increased in the presence of CsCl. The specific activities of the mutant enzymes were somewhat higher than the activity of the wild type ₂₂ complex at 37 °C (Table I) and at high temperatures (Fig. 1) in the presence of CsCl. The addition of GP stimulated the activities of the D56A or K167T enzymes in the presence of NaCl but inhibited the activities in the presence of CsCl. Although the activity of either mutant enzyme was lower in the presence of GP plus NaCl than in the presence of GP plus CsCl at low temperatures, the activities under the two conditions became similar at high temperatures (Fig. 1, B, C, E, and F).

View this table: [in this window] [in a new window]   Table I Effects of monovalent cations on the apparent activation energies and the activation entropies of the reaction rates of the tryptophan synthase 22 complex Conditions for activity measurements and data analysis by Arrhenius plots are given in Fig. 2 and in the text. Activity is specific activity in units/mg at 37 °C. Data for the wild type enzyme are from Ref. 1. WT, wild type.

The Arrhenius plots for the mutant enzymes were linear in the presence of NaCl or of GP plus CsCl but highly nonlinear in the presence of CsCl or GP plus NaCl (Fig. 1, E and F). The activation energies (E_a) and activation entropies (S) calculated from the Arrhenius plots are listed in Table I. The E_a value for either mutant enzyme in the presence of NaCl (141 or 122 kJ/mol) was similar to the E_a value for the wild type enzyme in the presence of NaCl at low temperature (128 kJ/mol). The E_a value for either mutant enzyme in the presence of CsCl at high temperature (~39 kJ/mol) was similar to the E_a for wild type enzyme in the presence of CsCl (34 kJ/mol). The E_a for the higher temperature portion of either mutant enzyme in the presence of NaCl and GP was similar to the E_a in the presence of CsCl and GP and was also similar to the E_a for the wild type enzyme in the presence of GP and NaCl or CsCl. The difference between the E_a and S values at low temperature and at high temperature calculated from nonlinear Arrhenius plots were also similar for the wild type and mutant enzymes (Table I).

The D56A and K167T Mutations Alter the Temperature Dependence of the Absorption Spectra of the ₂₂ Complex in the Presence of L-Serine and Other Ligands-- The conditions that yielded nonlinear Arrhenius plots (Fig. 1 and Table I) also yielded temperature-dependent absorption spectra for the wild type ₂₂ complex (1) and the D56A and K167T mutant enzymes (Fig. 2). The spectra obtained from the reactions of the mutant enzymes with L-serine in the presence of CsCl (Fig. 2, A and B) or in the presence of NaCl plus GP (Fig. 2, C and D) were strongly temperature-dependent and were very similar to those reported for the wild type ₂₂ complex in the presence of NaCl or GuHCl (1). Decreasing the temperature resulted in decreased absorbance at 340 nm (E-AA) and increased absorbance at 424 nm (E-Ser) (Scheme I). The presence of isosbestic points in the spectra (solid curves in Fig. 2, A-D) indicates that increasing the temperature shifted the equilibrium distribution of two intermediates from E-Ser to E-AA under these conditions. In contrast, the absorption spectra obtained from the reaction of either the D56A or the K167T ₂₂ complex with L-serine in the presence of NaCl exhibited a peak at 424 nm (dashed curves in Fig. 2, A and B), which decreased slightly with increasing temperature between 5 and 50 °C (Fig. 2, E and F). Thus, in the presence of NaCl, E-Ser was the predominant intermediate and temperature did not markedly alter the equilibrium distribution of the E-Ser and E-AA intermediates. The reactions of the mutant enzymes with L-serine in the presence of GP plus CsCl yielded spectra that had a peak at 340 nm (dotted curve in Fig. 2, C and D) and were essentially temperature-independent at 4-50 °C (Fig. 2, E and F). The results indicate that the combination of CsCl and GP stabilized the E-AA intermediate of the mutant ₂₂ complexes.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Effects of temperature on the absorption spectra of the D56A and K167T 22 complexes in the presence of L-serine. The absorption spectra of the D56A (A and C) or K167T (B and D) 22 complex (8 µM) were measured in the presence of 50 mM L-serine and either 0.1 M CsCl (A and B) or NaCl plus 50 mM GP (C and D) (solid lines). The spectra of the wild type 22 complex in the presence of NaCl or GuHCl exhibit similar temperature dependence (1). The spectra for either mutant enzyme were essentially temperature-independent in the presence of 0.1 M NaCl (dashed lines for data at 25 °C in A and B) or of 0.1 M CsCl + 50 mM GP (dotted lines for data at 25 °C in C and D). The absorbance at 424 nm from these spectra and from analogous experiments were plotted versus °C for the D56A (E) or K167T (F) 22 complex.

Analysis of the absorbance data for the reactions of the mutant enzymes with L-serine in the presence of CsCl or of NaCl plus GP and of the wild type enzyme with L-serine in the presence of NaCl (1) yielded the thermodynamic parameters (H_eq and S_eq) and T_m values for the conversion of E-Ser to E-AA (Table II). The H_eq and S_eq values were similar for the wild type and mutant enzymes (see "Discussion"). The T_m value for either mutant enzyme in the presence of CsCl (9 or 11 °C) was much lower than the T_m in the presence of NaCl and GP (18 or 21 °C). This result indicates that a higher temperature was needed to shift the equilibrium distribution of intermediates from E-Ser, which is favored by the open form, to E-AA, which is favored by the closed form, in the presence of GP and NaCl than in the presence of CsCl. Therefore CsCl, which binds at a site in the  subunit, was more effective in stabilizing the closed form than GP, which binds at the active site of the  subunit.

View this table: [in this window] [in a new window]   Table II Enthalpy and entropy changes for the equilibrium between E-Ser and E-AA Values of Heq, Seq, and Tm were obtained by analysis of the spectroscopic data for the wild type 22 complex (1) and for the mutant enzymes (Fig. 3). WT, wild type.

The D56A and K167T Mutations Alter the Temperature Dependence of Isotope Effects-- The conditions that yielded nonlinear Arrhenius plots (Fig. 1 and Table I) and temperature-dependent absorption spectra in the presence of L-serine (Fig. 2 and Table II) also yielded temperature-dependent changes in the kinetic isotope effects for the reaction of L-serine and indole for the wild type ₂₂ complex (1) and the D56A and K167T ₂₂ complex (Fig. 3). The primary kinetic isotope effects were small (~1.3) and essentially temperature-independent for the wild type ₂₂ complex in the presence of CsCl (1) and for the mutant enzymes in the presence of CsCl plus GP (Fig. 3). The kinetic isotope effect for either mutant enzyme in the presence of NaCl was large (~5) and changed only slightly with temperature. In contrast, the kinetic isotope effects in the presence CsCl or NaCl plus GP were temperature-dependent and decreased with increasing temperature, as observed with the wild type enzyme in the presence NaCl or GuHCl (1).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Temperature dependence of the kinetic isotope effects for the activities of the D56A (A) or K167T (B) 22 complex in the reaction with L-serine and indole. The primary kinetic isotope effect, which is the ratio of the activity with [-1H]DL-serine to the activity with [-2H]DL-serine, was plotted versus temperature. The wild type 22 complex exhibits similar temperature-dependent kinetic isotope effects in the presence of NaCl or GuHCl (1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previous investigations of the tryptophan synthase ₂₂ complex have shown that both the binding of a monovalent cation to a site in the  subunit and a salt bridge between Lys¹⁶⁷ and Asp⁵⁶ in the contact region between the  and  subunits (Scheme IIB) are important for the transmission of allosteric signals between the  and  subunits (8-10, 20).³ Our present results provide additional evidence that mutation of either Lys¹⁶⁷ or Asp⁵⁶ affects allosteric communication and catalysis, most likely by altering the conformational states of the  and  subunits. Our most important finding is that increased temperature combined with an  subunit ligand (GP) or a certain cation (Cs⁺ or NH₄⁺)⁵ reversed the deleterious effects of either mutation on activity. We propose that these activating conditions stabilize the active, closed conformation of the ₂₂ complex.

The occurrence of nonlinear Arrhenius plots under certain conditions (Fig. 2 and Table I) provides evidence for temperature-dependent conformational transitions in the wild type enzyme, as reported previously (1), and in the D56A and K167T ₂₂ complexes. A key finding is that there is an exact correlation between the effectors that yield nonlinear Arrhenius plots and the effectors that yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects for the wild type (1) and mutant ₂₂ complexes (Fig. 3 and Table III). Another important observation is that the differences between the values of E_a and S at low and high temperatures derived from nonlinear Arrhenius plots (Table I) were closely similar to the values of H_eq and S_eq for the conversion of E-Ser to E-AA obtained under analogous conditions (Table II) for both the wild type and mutant enzymes. These results support the proposal, made previously for the wild type enzyme (1), that the nonlinear Arrhenius plots result from conformational changes in the mutant enzymes. The absolute values of these thermodynamic constants were closely similar for both the wild type and the mutant enzymes, indicating that the same inferred conformational transition occurred with the wild type and mutant ₂₂ complexes. Stabilization of the higher activity, closed conformation of the mutant enzymes at both high and low temperatures required the combination of two stabilizing effectors (Cs⁺ and GP), whereas stabilization of the wild type enzyme required only one stabilizing effector (either Cs⁺ or GP) (Table I). This result provides evidence that the mutations shifted the equilibrium toward the more open conformation.

View this table: [in this window] [in a new window]   Table III Correlation between Arrhenius plots and temperature-dependent changes in absorption spectra and in primary kinetic isotope effects Abbreviation used: KIE, kinetic isotope effects; WT, wild type. Results for the wild type 22 complex are from Ref. 1.

The close parallel between the effects of mutation of either Lys¹⁶⁷ or Asp⁵⁶ on the temperature dependence data argues that interaction between these two residues is important for stabilization of the active, closed conformation of the  subunit. An analogous argument has been made on the basis of the similar effects of these two mutations on the reaction and substrate specificities (10).

We have previously discussed the thermodynamic parameters for the cases that yield nonlinear Arrhenius plots in relation to the simple reaction mechanism shown in Equation 6 (1).

(Eq. 6)

We showed (1) that at low temperatures, in the presence of Na⁺, the enzymatic rate constant k_cat = K_eq·k₃₁, where K_eq is the apparent steady-state equilibrium constant between E-Ser (open) and E-AA (closed) = k₂₃/(k₃₂ + k₃₁) in Equation 6, and k₃₁ is the rate constant of the rate-limiting step (E-Q₂  E-Trp). If these mutations have no effect on substrate binding or on interactions at the  site, the ~90-fold decreases in the specific activities of the mutant proteins, detected under these conditions, can be ascribed to similar decreases in K_eq. At 20 °C, disruption of the intersubunit salt bridge between Lys¹⁶⁷ and Asp⁵⁶ could destabilize the closed form of the protein by ~10.9 kJ/mol (i.e., RTln~90).

However, it is possible that each mutation also affects the conformation of the subunit in which it resides. Mutations of other  subunit residues that do not form salt bridges with  subunit residues have indeed been found to alter the substrate and reaction specificity (21-23). Our finding that the activity of the wild type ₂ subunit (1) was ~6-fold higher than that of the K167T ₂ subunit (data not shown) in the presence of NaCl and ~4-fold higher in the presence of CsCl at both high and low temperatures indicates that the conformation of the ₂ subunit is indeed altered by the K167T mutation. The present and earlier (9) findings that the deleterious effects of this mutation are repaired by association with the  subunit under some conditions show that the salt bridge is not essential for this intersubunit repair. Thus, activity measurements do not clearly distinguish between the effects of the K167T mutation on the conformation of the  subunit and on the effects on the salt bridge with D56A in the ₂₂ complex.

The small decreases in absorbance at 424 nm shown by the mutant complexes with L-serine at 40 °C in the presence of NaCl (Fig. 2, E and F) suggest that less than 5% of the enzymes are in the E-AA (closed) form. Under the same conditions, most of the wild type enzyme (>99%) is in this form (1). Such marked changes in the distribution of enzyme intermediates indicate a large reduction in K_eq (>2000-fold) for the mutant proteins, arising from destabilization of their closed form by at least 16.7 kJ/mol (i.e., RTln2000).

We next ask what information our results give on the importance of the Lys¹⁶⁷-Asp⁵⁶ salt bridge for allosteric communication? Important features of allosteric communication in the tryptophan synthase ₂₂ complex include mutual activation of the  and  subunits upon association and ligand-induced alterations of the equilibrium distribution of enzyme-substrate intermediates and of activity. The Lys¹⁶⁷-Asp⁵⁶ salt bridge is not essential for activation of the  subunit, as discussed above. Neither is the salt bridge essential for ligand-induced alterations in the distribution of intermediates, as shown by the effects of GP on the absorption spectra (Fig. 2). However, mutation of the salt bridge residues alters dramatically the effects of GP on the activity at the  site (Table I). The addition of GP increased the activity of the mutant enzymes in the presence of Na⁺. In contrast, GP reduced the specific activity of the wild type ₂₂ complex to about 14% in the presence of Cs⁺, but only reduced the activities of the mutant ₂₂ complexes under the same conditions to about 45% (Table I). We have suggested above that the activation of the mutant enzymes by GP in the presence of Na⁺ resulted from stabilization of the more active, closed conformation. The inhibition of the wild type enzyme by GP results from the stabilization of the closed form in which there is a decrease either in the rate constant of access of indole to the  site (24, 25) or in the rate-limiting step (E-Q₂  E-Trp in Scheme I). The smaller inhibition of the high activity form of the mutant enzymes by GP in the presence of Cs⁺ also suggests that the shift of the open to closed equilibrium is less complete in the mutant enzymes than in the wild type enzymes under these conditions. We conclude that D56 and K167 are not essential for catalysis and for allosteric communication between the  and  subunits but that mutual interaction is important in stabilization of the active, closed form of the ₂₂ complex.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Experimental Pathology, Holland Laboratory of American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.

^§ To whom reprint requests should be addressed: Section on Enzyme Structure and Function, Laboratory of Biochemistry and Genetics, Bldg. 8, Rm. 225, NIDDK, National Institutes of Health, 8 Center Dr. MSC 0830, Bethesda, MD 20892-0830. Tel.: 301-496-2763; Fax: 301-402-0240; E-mail, EdithM@intra.niddk.nih.gov.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001135200

² The term ₂ subunit is used for the isolated enzyme in solution;  subunit is used for the enzyme in the ₂₂ complex, for a specific residue in the  subunit or for the dissociation of the  and  subunits.

³ E. Woehl, O. Hur, D. Ferrari, C. Bagwell, U. Banik, L.-H. Yang, E. W. Miles, and M. F. Dunn, manuscript in preparation.

⁴ The turnover number, k_cat, is equal to a factor f times the specific activity for the ₂₂ complex (f = 0.00594).

⁵ The temperature dependence of the activity of the K167T mutant ₂₂ complex in the presence of NH₄Cl was closely similar to that in the presence of CsCl (data not shown).

	ABBREVIATIONS

The abbreviations used are: GP, DL--glycerol 3-phosphate; Bis-Tris, 1,3-bis[tris(hydroxymethyl)methylamino]; E-Ser, external aldimine of L-serine; E-AA, external aldimine of aminoacrylate; E-Trp, external aldimine of L-tryptophan; E-Q₁ and E-Q₂, quinonoid intermediates.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20302    most recent M001135200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fan, Y.-X. Articles by Miles, E. W. Search for Related Content PubMed