J Biol Chem, Vol. 274, Issue 44, 31189-31194, October 29, 1999

Catalytic Mechanism of the Tryptophan Synthase ₂₂ Complex
EFFECTS OF pH, ISOTOPIC SUBSTITUTION, AND ALLOSTERIC LIGANDS^*

Hyeon-Su Ro and Edith Wilson Miles^§

From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanism of the tryptophan synthase ₂₂ complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK_a1 = 6.5, pK_a2 = 7.3, and pK_a3 = 8.2-9. A significant primary isotope effect (~3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the -proton (Lys-87) is protonated at low pH, slowing the abstraction of the -proton and making this step at least partially rate-limiting. pK_a2 is assigned to Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK_i values of 7.3. The residue with this pK_a (Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK_a values support the assignment of pK_a2 to Lys-87 and suggest that the ionizing residue with pK_a1 could be a carboxylate, possibly Asp-305, and that the residue associated with a conformational change at pK_a3 may be Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The tryptophan synthase ₂₂ complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ₂ subunit¹ and the  subunit in the ₂₂ complex catalyze the pyridoxal phosphate (PLP)² -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion).

The three-dimensional structure of the tryptophan synthase ₂₂ complex from Salmonella typhimurium (5) revealed that the active sites of the  and  subunits are ~25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the  subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the  subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the  and  sites. These heterotrophic interactions are proposed to switch the  and  subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the  subunit and of a "mobile region" (6) or "communication domain" (7) in the  subunit. These results support the proposal that the abstracted -proton of L-serine is sequestered in a solvent-excluded site (8).

Investigations of the pH dependence of enzyme reactions can give information on enzyme mechanism and on the identity of residues that play catalytic and regulatory roles (9-11). Here we report the effects of pH, of temperature, of isotopic substitution, and an allosteric ligand (DL--glycerol 3-phosphate) on the PLP-dependent reaction of the tryptophan synthase ₂₂ complex with L-serine and indole to form L-tryptophan. The results indicate that three ionizing groups are involved in substrate binding and catalysis with pK_a1 = 6.5, pK_a2 = 7.3, and pK_a3 = 8.2-9. Correlation of the kinetic results with structural and mutational data suggests that the ionizing residues are Asp-305, Lys-87, and Lys-167.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Buffer-- PLP, pyridoxal hydrochloride, L-serine, and DL--glycerol 3-phosphate were purchased from Sigma. Oxindolyl-L-alanine, which was a generous gift of Robert S. Phillips, was synthesized by the method of Savige and Fontana (12) as described (13). MOPS and proline were from Fluka. Bicine was from United States Biochemical. DL-[-²H]serine was prepared as reported (14). MBP buffer (15) containing 50 mM MOPS, 50 mM Bicine, 50 mM proline, and 1 mM EDTA was used for kinetic and spectroscopic studies with additional 100 mM NaCl. The pH was raised with NaOH to pH 11.2; the solution was then back-titrated with HCl to the desired pH value.

Bacterial Strain, Plasmid, Enzyme Preparation, and Enzyme Assay-- The plasmid pEBA-10 (16) was used to express the wild type tryptophan synthase ₂₂ complex from S. typhimurium in E. coli CB 149 (17), which lacks the trp operon. The ₂₂ complex was purified by a method using crystallization (18). Protein concentrations were determined from the specific absorbance at 278 nm using A = 6.0 for the ₂₂ complex (19). Activity of tryptophan synthase in the -replacement reaction with L-serine and indole ( reaction) was determined by a spectrophotometric assay (19) in the presence of 100 mM NaCl and L-[-¹H]serine or DL-[-²H]serine. Because the D-isomer of Ser is unreactive with tryptophan synthase (8, 14, 20, 21), DL-Ser can be used in place of L-Ser for investigations of the catalytic mechanism. DL--Glycerol 3-phosphate (50 mM) was added where indicated. One unit of activity in any reaction is the formation of 0.1 µmol of product in 20 min at 37 °C. We demonstrated that the tryptophan synthase ₂₂ complex is stable in the pH range of 6.5-9.6 by preincubating the enzyme at pH values over this range and then determining activity at pH 7.8; the activity was not changed by the preincubation at any pH in this range (data not shown).

Data Analysis-- Initial reaction rates in the reaction of L-serine and indole to form L-tryptophan were obtained as a function of L-serine concentration and of pH in the presence of 200 µM indole. The data were analyzed by published methods (10, 11, 22, 23). The kinetic parameters (V, K)² were determined by fitting the initial rate data to Equation 1.

(Eq. 1)

The values of V and K are apparent values because indole was not saturating. It is not possible to use saturating indole concentrations because the ₂₂ complex is inhibited by concentrations of indole greater than 30 µM (24). Because the ₂ subunit alone is not inhibited by indole, Heilmann (24) concluded that indole inhibits the ₂₂ complex by binding to 1 or 2 indole sites in the  subunit. The concentration of indole in our assays, 200 µM, is 4-27 times higher than the K_m values that have been reported at pH ~7.8 for the ₂₂ complex from E. coli (13 µM (25), 50 µM (24), and 7.4 µM (26)) and for the ₂₂ complex from S. typhimurium (18 µM (27)). Lane and Kirschner have reported that the K_m for indole decreased from 42 µM at pH 6.1 to 8.0 at pH 8.0. Thus, the concentration of indole in our assays is higher than the K_m for indole even at low pH.

Data for competitive inhibition was fitted to Equation 2,

(Eq. 2)

where K is the Michaelis constant, K_i is the inhibition constant of a competitive inhibitor, and [I] is the concentration of the competitive inhibitor. pK_i profiles for glycine and oxindolylalanine were fitted to Equation 3 (see Equation 57 in Ref. 10) because pH profiles are level above single pK but decrease with a +1 unit slope below the pK.

(Eq. 3)

When the logV profile was bell-shaped with a slope of +1 at the acidic side and 1 at the basic side, the data were fitted to Equation 4 (see Equation 27 in Ref. 22, and Equation 11 in Ref. 23).

(Eq. 4)

When the logV/K profile exhibited a slope of +2 at the acidic side and 1 at the basic side, the data were fitted to Equation 5 (see Equation 12 in Ref. 23).

(Eq. 5)

When pK_a1 and pK_a2 values were too close to give separate pK_a values, the data were fitted to Equation 5 defining pK_a1 = pK_a2. In Equations 3-5, pK_i*, logV*, and log(V/K)* are the pH-independent values of the parameters, and K₁, K₂, and K₃ are dissociation constants for enzyme groups. The enthalpy of ionization (H_ion) was determined by fitting the pK values obtained at different temperatures to Equation 6,

(Eq. 6)

where H_ion, T, and R are the enthalpy of ionization, the absolute temperature, and the gas constant (1.987 cal/mol/deg), respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

pH Dependence of the Kinetic Parameters-- The effect of pH on the conversion of L-serine and indole to L-tryptophan by the tryptophan synthase ₂₂ complex was determined over the pH range of 6.0-9.5 while varying L-serine concentrations at a fixed level of indole (0.2 mM). This concentration of indole gives the highest rate at each pH value and is greater than the K_m for indole (see data analysis under "Experimental Procedures"). Higher concentrations of indole are inhibitory (24). As seen in Fig. 1A, the data for log V fit well using Equation 4 to a bell-shaped curve with slopes of +1 and 1 on the acidic and basic sides, respectively, indicating dependence on two ionizing groups with pK_a values of ~6.5 and ~8.2 (line 1 in Table I). Data for the pH dependence of log V/K (Fig. 1A) fit best using Equation 5 to a bell-shaped curve with slopes of +2 and 1 on the acidic and basic sides, respectively, indicating dependence on three ionizing groups with pK_a1,2 = ~7.0 and pK_a3 = ~9.0 (line 2 in Table I). A value of pK_a2 = ~7.3 (line 3 in Table I) could be calculated from the fit of the same data to Equation 5 assuming a value of pK_a1 = 6.5 from the temperature dependence of pK_a1 (see below).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   The pH dependence of log V () and log V/K () for the L-serine + indole  L-tryptophan + H2O reaction of the tryptophan synthase 22 complex. Data in absence (A) or presence (B) of DL--glycerol 3-phosphate (GP, 50 mM). The slopes of the acidic and basic sides of the plots derived from fits of the data are indicated by dashed lines.

View this table: [in this window] [in a new window]   Table I pKa values associated with tryptophan synthase Initial rates in the reaction of L-serine + indole  L-tryptophan + H2O, varying L-serine concentration at 37 °C in the presence of 0.2 mM indole (Figs. 1 and 3) and in the presence or absence of 50 mM DL--glycerol 3-phosphate (GP). The pH profiles of logKi for oxindolyl-L-alanine (Oxa) or glycine (Gly) were derived from plots of competitive inhibition versus L-serine (Fig. 3).

Effects of an Allosteric Ligand on the pH Dependence of the Kinetic Parameters-- The substrate analog DL--glycerol 3-phosphate binds to the active site of the  subunit and stabilizes the closed conformation of the ₂₂ complex (15). The pH profile for log V in the presence of DL--glycerol 3-phosphate (Fig. 1B) decreases below an acidic pK_a = 6.8 and above a basic pK_a = 8.8 (Table I). The acidic side of the log V/K profile exhibits a slope of +1 and yields a pK_a = 7.3; the basic side yields a pK_a of 8.7 (Table I).

Effect of Temperature on pK_a Values-- The temperature dependence of log V and log V/K was determined to investigate the nature of groups whose ionization state affect the activity of tryptophan synthase. Activities were measured at 15, 25, and 37 °C, and the data were fit to Equations 4 or 5 to give pH profiles of log V (Fig. 2A) and log V/K (Fig. 2B). The derived pK_a values are listed in Table II. The values of pK_a1 and pK_a2 in the log V/K profile were sufficiently separated to be calculated at 15 and 25 °C but not at 37 °C. Because the values of pK_a1 appeared to be temperature-independent and equal to ~6.5 at all three temperatures in the log V profile and at 15 and 25 °C in the log V/K profile, the value of pK_a1 for log V/K at 37 °C was assumed to be 6.5 as well. This value was used in Equation 5 to calculate pK_a2 (Table II and Fig. 2). From plots of the values of pK_a versus 1/T (Fig. 2, C and D), lines were obtained and analyzed by using Equation 6 to yield values for H_ion, the enthalpy of ionization (Table II).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of temperature on the pH dependence of log V and log V/K for the L-serine + indole  L-tryptophan reaction. A, logV; B, log V/K; Arrhenius plots of pKa values derived from analysis of data for log V (C) and log V/K (D).

View this table: [in this window] [in a new window]   Table II pKa values at various temperatures and derived Hion values Initial rates in the reaction of L-serine + indole  L-tryptophan + H2O, varying L-serine concentration at 15, 25, and 37 °C for determination of Hion (Fig. 2).

pH Dependence of Inhibition by Substrate Analogs-- To determine whether pK_a values observed in the log V/K profile were true or apparent values, we measured the effect of pH on inhibition by substrate analogs. Values of pK_i were determined from plots of competitive inhibition by glycine (Fig. 3A) and by oxindolyl-L-alanine (data not shown) versus L-serine in the -replacement reaction with L-serine and indole over a range of pH values from 6-9.5. The pH profile for pK_i for each inhibitor was a half-bell with a limiting slope of +1 on the acidic side (Fig. 3B). The data were fit to Equation 3 to yield pK_a values of ~7.3 for glycine and for oxindolyl-L-alanine; the pH-independent values of K_i are ~0.1 M for glycine and 0.05 mM for oxindolyl-L-alanine (Table I).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Dependence of pKi for glycine and oxindolyl-L-alanine (oxa) on pH (B) derived from plots of competitive inhibition versus L-serine at pH 7.8 (A) and from analogous plots at other pH values (data not shown).

pH Dependence of Deuterium Isotope Effects-- The primary deuterium isotope effects for abstraction of the -proton of L-serine were measured as a function of pH (Fig. 4). Both ^DV and ^D(V/K)² are pH-dependent and become negligible (i.e. ~1) above pH 9. Both of these effects increase and become equal below pH 7, ^DV = 3.5 and ^D(V/K) = 3.5. 

View larger version (13K): [in this window] [in a new window]   Fig. 4.   pH dependence of the primary deuterium kinetic isotope effects on V (DV)() and D(V/K) ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The reaction of L-serine and indole to form L-tryptophan proceeds through a series of PLP intermediates at the active site of the  subunit in the tryptophan synthase ₂₂ complex (Scheme I) (13, 20, 28-30). Similar mechanisms are utilized by other PLP enzymes that catalyze -elimination and -replacement reactions (28, 31), including O-acetylserine sulfhydrylase (32, 33), tryptophan indole lyase (34), and tyrosine phenol lyase (35). The reactions in Scheme I include the release and transfer of three different protons. The reaction of the protonated internal aldimine (E) with the protonated substrate, L-serine, to form ES-I yields an extra proton, shown in a triangle, which may be used subsequently to protonate the leaving hydroxyl group of L-serine in the conversion of ES-III to ES-IV. The -proton of L-serine, shown in a square, is abstracted by Lys-87 in the conversion of ES-II to ES-III and is subsequently used to protonate the quinonoid ES-VI to form ES-VII. The C-3 proton of indole, shown in a circle, is removed during the tautomerization of the indolenine intermediate (ES-V to ES-VI) and may be used to protonate the product, L-tryptophan, as it is released.

View larger version (27K): [in this window] [in a new window]   Scheme 1.   Intermediates and proton transfers in the reaction of tryptophan synthase (E) with L-serine and indole to form L-tryptophan and H2O.

Further elucidation of the mechanism of the reaction in Scheme I would be facilitated by identifying residues in tryptophan synthase that catalyze the proton transfer steps and by identifying the pK_a values of these residues. Investigations of the pH dependence and temperature dependence of kinetic parameters have frequently been used to deduce the intrinsic pK_a values of functional groups in enzymes. A systematic investigation of these factors is an important step in determining an enzyme mechanism. Although the identification of specific residues and their pK_a values may sometimes be problematic (9), the kinetic results combined with information from structural investigations and site-directed mutagenesis can yield important support for a proposed mechanism.

The pH profiles for the steady-state kinetic parameters of tryptophan synthase show that three ionizing groups are important for substrate binding and catalysis (Fig. 1A and Table I). The group with pK_a2 = ~7.3 appears in the log V/K profile but not in the log V profile, indicating that this group must be in the correct protonation state for the substrate to bind. The pH profiles of pK_i for glycine and for oxindolyl-L-alanine, which are competitive inhibitors of L-serine, show that a single residue with a pK_a value of 7.3 must be unprotonated for binding glycine or oxindolyl-L-alanine and, by inference, L-serine (Fig. 3B and Table I). The coincidence of this pK_a value with pK_a2 in the log V/K profile shows that pK_a2 is the correct pK_a value for the ionizing group and rules out drastic stickiness of the substrate (10). A sticky substrate, which reacts to give products more rapidly than it dissociates from the enzyme, gives incorrect pK_a values that differ from the pK_a values obtained for inhibitors. Because L-serine, glycine, and oxindolyl-L-alanine have no ionizable groups in the range of pH measured, the observed pK_a values reflect enzyme groups that must be deprotonated or protonated for maximum binding and catalysis.

Oxindolyl-L-alanine, which has tetrahedral geometry at C-3 of the indole ring, is a structural analog of the indolenine tautomer of L-tryptophan, which is a proposed intermediate in reactions of tryptophan synthase (E-V in Scheme I) and tryptophan indole-lyase (13, 29). The finding that oxindolyl-L-alanine is a potent competitive inhibitor of both enzymes provided strong evidence for the intermediacy of the indolenine tautomer of L-tryptophan in reactions catalyzed (13, 29). The K_i value (0.05 mM) found for oxindolyl-L-alanine at pH 7.8 in the present work (Fig. 3) is about 10-fold higher than that reported previously (13). However, the previous K_i value was obtained from assays of the reaction of L-serine with indole-3-glycerol phosphate, a substrate that binds to the  subunit and is known to increase the affinity of ligands that bind to the  subunit (17).

The pH profiles of pK_i for oxindolyl-L-alanine and for glycine each show a requirement for a single group with a pK_a of ~7.3 to be unprotonated (Fig. 3). In contrast, analogous studies with tryptophan indole-lyase showed a requirement for two groups to be unprotonated for the binding of oxindolyl-L-alanine with pK_a values of 6.0 and 7.6 and for one group to be unprotonated for the binding of alanine with a pK_a value of 7.6 (34). The results for tryptophan indole-lyase suggested that the group with the pK_a of 6.0 abstracted the ring nitrogen proton, facilitating the formation and stabilization of the indolenine tautomer of tryptophan. The absence of a second pK_a for oxindolyl-L-alanine binding to tryptophan synthase indicates that the group with pK_a1 is not involved in binding this analog.

Addition of an  subunit ligand, DL--glycerol 3-phosphate, increases the values of pK_a1 and pK_a3 in the log V profile (Fig. 1B and Table I). Stabilization of the closed conformation of ₂₂ complex by the  subunit ligand may increase these pK_a values by increasing the hydrophobicity of the environment of these two groups. The reason for the absence of a third ionizing group corresponding to pK_a1 in the V/K profile is unknown.

Our most important results are the pH dependence of the primary deuterium isotope effects of abstraction of the -proton of L-serine (^DV and ^D(V/K))² (Fig. 4). Both of these effects increase with decreasing pH and become essentially equal (~3.5) below pH 6.8. The significant primary kinetic isotope effect at low pH indicates that the general base has been protonated, slowing the abstraction of the -proton and making this step at least partially rate-limiting. This permits the highly probable assignment of one of the pK_as to the base that accepts the -proton.

An alternative interpretation of the kinetic isotope effect results is that another step becomes rate-limiting at higher pH. Knowles (9) has discussed this problem and quoted Jencks (51) as pointing out that a "mirage" pK_a may be seen if the elementary step affected by the ionization is not rate-determining at all pH values. This type of pK_a has also been termed a "kinetic pK_a" (36). This alternative interpretation is supported by our conclusion that the rate of the reaction of L-serine with indole decreases at high pH as the result of a conformational change that produces an open form in which removal of the hydroxyl group of L-serine becomes rate-limiting (see below). In contrast, the rate of reaction of -chloro-L-alanine with indole increases almost linearly between pH 7 and 9 (data not shown), consistent with evidence that -elimination of the chloride-leaving group of -chloro-L-alanine is not rate-limiting in the open conformation (37).

Crystallographic studies (5-7, 38) have revealed the presence of several ionizing residues in the substrate binding site of the  subunit: Lys-87, His-86, His-115, Asp-305, and Glu-109. The identity of Lys-87 as the base that accepts the -proton of L-serine is strongly supported by both x-ray crystallography (6, 39) and by mutagenesis studies (40).

Assignment of pK_a2-- Several types of evidence support the assignment of pK_a2 = ~7.3 to Lys-87. First, the primary kinetic isotope effects for ^DV and ^DV/K decrease above pH 7 (Fig. 4). Second, this pK_a value is observed for inhibition by glycine and oxindolyl-L-alanine and not in the logV plot (Figs. 1 and 3 and Table I), indicating that this ionizing group must be unprotonated for binding L-serine, glycine, or oxindolyl-L-alanine. These amino acids, which are largely protonated in the neutral pH range, would be expected to bind to the unprotonated Lys-87. Third, the thermal dependence of pK_a2 is characteristic of a lysyl residue (Table II). Fourth, mutation of His-86 (H86L) alters the pH profiles of absorbance and fluorescence signals and shifts the pH optimum for the synthesis of L-tryptophan from pH 7.5 to pH 8.8.³ These new results provide evidence that His-86 facilitates catalysis in the physiological pH range by decreasing the pK_a of the catalytic Lys-87 in the wild type ₂₂ complex.³ They also provide a structural explanation for the low pK_a value assigned here to Lys-87.³ Fifth, very similar pH profiles for ^DV and ^D(V/K) were observed for tyrosine phenol-lyase from Erwinia herbicola (35) and led to the assignment of a pK_a = ~7.6-7.8 to the catalytic lysyl residue of tyrosine phenol-lyase that abstracts the -proton of L-tyrosine.

Previous investigations of tryptophan synthase have demonstrated a primary kinetic isotope effect for the L-serine deaminase reaction of the ₂ subunit at pH 7.8 (14), for the synthesis of L-tryptophan (20, 41, 42), and for the equilibrium distribution of intermediates in the reaction of L-serine (8) and in the reaction of L-serine and indole (26).

Assignment of pK_a1-- Our finding that pK_a1 = ~6.5 is essentially temperature-independent (Fig. 2 and Table II) suggests that this residue is a neutral acid, e.g. a carboxylate (10). Although the validity of the use of H_ion values for deducing the nature of ionizing groups has been criticized (9), the assignment of a pK_a = 6.5 to a carboxylate in a hydrophobic site seems not unreasonable. Although Asp-305 and Glu-109 are both located in the active site of the  subunit (6, 7, 38), Glu-109 is close to the indole nitrogen of L-tryptophan in the ES-VIII intermediate (Scheme I) (6) and appears too distant to accept a proton. The crystal structure of the external aldimine of L-serine (ES-I in Scheme I) formed by a mutant enzyme (K87T) suggests that the interaction of the carboxylate of Asp-305 with the hydroxyl of L-serine is important to the chemistry of -elimination to yield ES-II (6). Consequently, Asp-305 is the most likely candidate for the residue with pK_a1 = ~6.5 that accepts the initial proton, indicated by the triangle, and protonates the hydroxyl-leaving group of L-serine.

Assignment of pK_a3-- The group with pK_a3 = 8.2 in the log V profile and = 9 in the log V/K profile (Fig. 1 and Table I) must be protonated for substrate binding and catalysis because the pH dependence of log V/K follows ionizations in the free enzyme and the free substrate (36). V/K contains all steps up to and including the first irreversible step, which is addition of indole to ES-IV. The group with pK_a3 = 8-9 exhibits a H_ion of 17-20 kcal/mol (Table II), suggesting that this ionization is that of a protein residue which is linked to a conformational change (10) and consequently is not that of the amino group of the substrate L-serine (pK_a = 9). The observed pK_a3 = 8-9 is probably not the true pK_a of this ionizing residue because the observed change in free energy is the sum of the free energy of ionization and the free energy of the conformational change.

We propose that this conformational change is the conversion of the closed form of the tryptophan synthase ₂₂ complex to the open form (for a review, see Ref. 4). This proposal is supported by our finding that log V/K shows no pK_a3 in the presence of Cs⁺, a cation that stabilizes the closed conformation (43) (data not shown). We have recently observed that activity in the -elimination reaction (L-serine  pyruvate and NH₃) increases nearly linearly between pH 6.7 and 9.³ This result suggests that the open conformation of the ₂₂ complex is formed at high pH and that the aminoacrylate intermediate (ES-IV in Scheme I) is more accessible to hydrolysis by solvent water in the open form.³ Increased -elimination activity is also exhibited by the open form of the enzyme that is promoted by mutation, solvents, or low concentrations of urea or guanidine hydrochloride (37, 43-48). A previous investigation has also provided evidence for a pH-dependent conversion of the ₂₂ complex from a closed conformation at low pH to an open conformation at high pH (15).

Crystallographic studies (6, 49) have identified several ionizing residues that may be important for stabilizing the active, closed conformation of the ₂₂ complex: Arg-141, Arg-148, Lys-167, Arg-175, and Lys-382. The most likely candidate is Lys-167 which forms an ion pair with Asp-56 in the closed conformation. Mutation of this residue favors the open conformation and impedes communication between the  and  subunits (44, 50).

In conclusion, our studies of the temperature and pH dependence of kinetic parameters and of the effects of isotopic substitution provide evidence that three ionizing groups are involved in substrate binding and catalysis by the tryptophan synthase ₂₂ complex. The residue with pK_a = 6.5 is likely a carboxylate that accepts a proton from the -amino group of L-serine and then uses the proton to promote the dehydration of L-serine. Asp-305 is a good candidate to serve this role. The residue with pK_a = 7.3 must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. The pH dependence of the primary isotope effects suggests that this residue is Lys-87 which accepts the -proton of L-serine and then uses the proton to protonate the quinonoid of L-tryptophan. Ionization of a residue linked to a conformational change (pK_a3 = ~9) destabilizes the active, closed form of the enzyme. Lys-167 is a good candidate to play this role.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Peter McPhie and Dr. Sangkee Rhee for helpful discussions and comments on the manuscript.

	FOOTNOTES

^* A preliminary report of portions of this work was presented at the American Society for Biochemistry and Molecular Biology, May 16-20, 1999, in San Francisco, CA.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: Laboratory of Metabolism, National Cancer Institute, Bldg. 37, Room 3D25, Bethesda, MD 20892.

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

¹ The term ₂ subunit is used for the isolated enzyme in solution;  subunit is used for the enzyme in the ₂₂ complex or to describe a specific residue in the  subunit.

³ Ro, H.-S., and Miles, E. W. (1999) J. Biol. Chem. 274, in press.

	ABBREVIATIONS

The abbreviations used are: PLP, pyridoxal phosphate; Bicine, N,N-bis(2-hydroxyethyl)glycine; MOPS, 3-(N-morpholino)propanesulfonic acid; MBP, MOPS, Bicine, proline; H_ion, H_ionization; V, apparent V_max; K, apparent K_Ser; ^DV, apparent V_max (L-[-¹H]serine)/apparent V_max (-DL-[-²H]serine; ^D(V/K), apparent V_max/K_ser (L-[-¹H]serine)/apparent V_max/K_Ser (DL-[-²H]serine). See data analysis under "Experimental Procedures" for information relevant to apparent V_max and apparent K_Ser values.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES