©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Monovalent Cations Partially Repair a Conformational Defect in a Mutant Tryptophan Synthase Complex (-E109A) (*)

(Received for publication, March 10, 1995; and in revised form, May 15, 1995)

Sergei B. Ruvinov S. Ashraf Ahmed Peter McPhie Edith Wilson Miles (§)

From the Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We are using the tryptophan synthase complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the subunit alone is quite low, but is activated by certain monovalent cations or by association with subunit to form an complex. Since monovalent cations and subunit appear to stabilize an active conformation of the subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of complex having subunit glutamic acid 109 replaced by alanine (E109A). The E109A complex is inactive in reactions with L-serine but active in reactions with -chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type subunit and complex. We find that CsCl stimulates the activity of the E109A complex and of wild type subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the complex by stabilizing a conformation with catalytic properties more similar to those of the wild type complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate-dependent enzymes by stabilizing alternative conformations.


INTRODUCTION

The regulation of enzyme activity and of substrate specificity is a problem of critical importance in biology. Enzyme activity can be modulated by interaction of the enzyme with ligands or with other proteins. These interactions produce changes in protein conformation or alter the equilibrium distribution of preexisting conformations. Mutations can also modulate enzyme activity by altering protein conformation. In the present work we ask whether ligands that stabilize the active conformation of an enzyme can also repair the deleterious effects of a mutation that leads to an inactive conformation.

Tryptophan synthase (EC 4.2.1.20) is a classic system for investigating how ligands, protein-protein interaction, and mutations regulate enzyme activity and substrate specificity (for reviews, see (1, 2, 3, 4, 5) ). The bacterial enzyme is an complex that dissociates reversibly into monomeric subunits and dimeric subunits. The separate subunit catalyzes the reversible aldolytic cleavage of indole-3-glycerol phosphate to D-glyceraldehyde 3-phosphate and indole, termed the reaction. The separate subunit catalyzes the condensation of L-serine with indole to form L-tryptophan in a pyridoxal phosphate-dependent -replacement reaction, termed the reaction. The interaction of the and subunits in the complex serves to increase the rates of the and reactions up to 100-fold, increase substrate binding affinities, and alter reaction specificities. These changes are attributed to conformational changes which occur upon assembly of the complex(6) . The binding of a ligand or the formation of a reaction intermediate at the active site of either subunit in the complex alters the reaction kinetics at the heterologous active site 25 Å distant(7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . The combined results suggest that the and subunits undergo conformational changes during catalysis and exist in two or more conformations that have been designated ``open'' and ``closed'' (11) .

Investigations of the reaction and substrate specificities of wild type and mutant forms of the subunit and the complex also provide evidence that the subunit can exist in two different conformational states (designated I and II in Table 1)(17, 18) . The separate subunit catalyzes -replacement and -elimination reactions with a variety of amino acids that have an electronegative substituent in the -position (e.g.L-serine and -chloro-L-alanine)(19, 20) .

-replacement reaction:

-elimination reaction:



Association of the subunit with the subunit greatly increases activity in the -replacement reaction with L-serine and indole and almost completely eliminates activity in the -elimination reaction with L-serine. Thus association with the subunit alters the reaction specificity of the subunit. Whereas L-serine is a better substrate than -chloro-L-alanine for the -replacement reaction with indole catalyzed by the complex, the reverse is true for the subunit(17) . Thus association with the subunit alters the substrate specificity of the subunit. Several complexes with single amino acid replacements in the active site of the subunit have substrate and reaction specificities similar to those of the wild type subunit(17) . This finding led us to suggest that the wild type subunit and these mutant complexes exist in open conformations (I in Table 1), whereas the wild type complex exists in a closed conformation (II in Table 1)(17) . Aliphatic alcohols also stabilize an alternative conformation of the complex that has properties similar to that of the free subunit in aqueous solution (21) (I in Table 1).

Early studies showed that some monovalent cations (K, Li, and NH, but not Na) activate the subunit (22, 23) in -replacement and -elimination reactions with L-serine. High concentrations of NH in some ways mimic the effects of the subunit on the kinetic and spectroscopic properties of the subunit(24, 25, 26) . Recently it has been shown that monovalent cations (Na, K, Li, Rb, NH, and Cs) activate the wild type tryptophan synthase complex and alter the pH-dependent equilibrium distribution of enzyme-substrate intermediates(27, 28, 29, 30, 31) . Together the results support the idea that both the free subunit and the complex exist in two or more conformations. Preferential binding of certain monovalent cations to a specific site in the subunit may alter the equilibrium distribution of preexisting conformations in the subunit and complex by stabilizing conformation II (Table 1). Binding of an subunit ligand (-glycerol 3-phosphate) to the complex also alters the distribution of intermediates formed with L-serine(32) . Table 1indicates that site ligands stabilize conformation II.

Because certain cations and site ligands appear to stabilize the more active conformation II of wild type subunit and complex (Table 1), we reasoned that these ligands might promote the conversion of a mutant complex from an inactive conformation I to an active conformation II. In the present work we have investigated the effects of monovalent cations on a mutant complex having Glu-109 of the subunit replaced by alanine (E109A). Glu-109 is located in a region of the subunit adjacent to the covalently bound pyridoxal phosphate cofactor and near the putative indole-binding site of the subunit (see Fig. 1in (17) ). It has been postulated that the carboxylate of Glu-109 may activate the indole toward nucleophilic attack on the aminoacrylate (33) and/or activation of the -hydroxyl of L-serine as a leaving group(18) . The finding that the E109A complex can synthesize L-tryptophan from -chloro-L-alanine and indole but not from L-serine and indole provides evidence that Glu-109 is not an essential residue for the activation of indole but may serve to activate the hydroxyl of L-serine as a leaving group(17, 18, 34) . Alternatively, replacement of Glu-109 by alanine (17) or aspartate (16, 18) may introduce structural changes within the subunit that alter the conformation of the complex and thereby alter the substrate and reaction specificity. Our finding that the E109A complex resembles the wild type subunit in substrate specificity and reaction specificity suggests that the mutation stabilizes conformation I (see ``mutations'' in Table 1). The results reported here show that addition of a high concentration of CsCl partially restores the activity of the E109A complex with L-serine and alters the substrate and reaction specificity. The results suggest that CsCl repairs a conformational defect in the E109A complex by stabilizing conformation II that has structural and catalytic properties more similar to those of the wild type complex.


Figure 1: Effects of CsCl concentration on activity in the -replacement reaction with L-serine and indole. A, wild type complex - GP () or + 10 mM GP (); B, wild type subunit (); C, E109A complex - GP () or + 10 mM GP ().




EXPERIMENTAL PROCEDURES

Chemicals and Buffers

DL--Glycerol 3-phosphate (GP),()-chloro-L-alanine (hydrochloride), and pyridoxal phosphate were from Sigma. All spectroscopic experiments and enzyme assays utilized Buffer B (50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8).

Enzymes

Wild type (35) and E109A (36) forms of the tryptophan synthase complex and wild type subunits (37) from Salmonella typhimurium were isolated and purified to homogeneity. Protein concentrations were determined from the specific absorbance at 278 nm of the complex (E = 6.0) or of the subunit (E = 6.5)(38) .

Enzyme Assays

One unit of activity in any reaction is the formation of 0.1 µmol of product in 20 min at 37 °C. -Replacement reactions with L-serine or -chloro-L-alanine and indole were measured by a direct spectrophotometric assay (38) containing modified components (100 mML-serine or 40 mM -chloro-L-alanine, respectively, and 0.2 mM indole in Buffer B).

Spectroscopic and Analytical Methods

Absorption spectra were made using a Hewlett-Packard 8452 diode array spectrophotometer. Time course measurements of enzymatic activities at single wavelengths were made using a Cary 118 spectrophotometer.


RESULTS

The results reported here were all carried out in 50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8, since we had used this buffer for previous related experiments(17) . Although Na does not activate the separate subunit(22, 23) , recent studies show that Na does activate the wild type complex(27, 28, 29, 30, 31) . These recent studies utilized triethanolamine and bis-tris propane buffers that contain no monovalent cations and are thus better suited for studies of the effects of cations. Because Na in our buffer (50 mM) may compete with other cations for the same binding site, our results do not show the effects of the absolute concentrations of other cations. However, our results do show that other cations have striking effects on the properties of the wild type subunit and complex and on the E109A complex.

Effects of Monovalent Cations on Activity

Table 2shows the effects of various monovalent cations as their chlorides (0.2 M) on the specific activity of the wild type subunit in the -replacement reactions with L-serine and indole and with -chloro-L-alanine and indole. Addition of Li or NH significantly stimulates activity with L-serine and indole, as found previously with partially purified subunit from Escherichia coli(23) . Stimulation of the subunit by Rb and Cs is reported for the first time. Because Cs gave the greatest stimulation, Cs was selected for further study. The monovalent cations have much smaller effects on the activities with -chloro-L-alanine plus indole. Thus the monovalent cations that increase the activity with L-serine and indole alter the substrate specificity by increasing the ratio of activity with L-serine to activity with -chloro-L-alanine (see serine:-chloro-L-alanine activity ratio in Table 2). Note that the ratio of activities with L-serine and -chloro-L-alanine is one of the features that distinguish the postulated conformations I and II (Table 1).



Fig. 1shows the effects of CsCl concentration on activity in the -replacement reaction with L-serine and indole. Low concentrations of CsCl activate the wild type complex (A) and subunit (B). High concentrations of CsCl activate the E109A complex (C) but inhibit the wild type complex (A). The subunit ligand, GP, alters the effect of CsCl concentration on the activities of the wild type and E109A complexes.

The activity data in Fig. 1can be represented by a model (see Table 3) which assumes the noncooperative binding of CsCl to one or two classes of sites on the protein with average dissociation constants K and K. The binding of CsCl converts the enzyme from the CsCl free form X to CsCl-bound forms Y or Z, with concomitant changes in activity. Addition of higher concentrations of CsCl (100-3000 mM) to the wild type complex (Fig. 1A) produces further changes of the activity which vary monotonically with salt concentration. These changes could result from additional binding at very weak sites on the protein or from the non-ideal behavior of concentrated salt solutions. For the purpose of curve fitting, these changes are expressed as a linear function of salt concentration and described as a ``solvent effect'' in Table 3. The curves shown in Fig. 1, A-C, were the best fits to this model. Table 3describes the model and the derived dissociation constants. The results give no indication of the stoichiometry of the reaction between CsCl and the enzyme.



Effects of CsCl on Absorption Spectra

The absorption spectra of the wild type complex (Fig. 2A), subunit (Fig. 2B), and E109A complex (Fig. 2C) are very similar in the absence of L-serine. Each enzyme exhibits a major peak centered at 412 nm due to the internal aldimine formed between pyridoxal phosphate and subunit Lys-87 (E in Fig. SI). Reaction of the wild type complex with L-serine in the presence or absence of CsCl yields a complex spectrum with a major peak centered at 340 nm (Fig. 2A), which is ascribed to the pyridoxal phosphate-amino acrylate Schiff base (E-AA in Fig. SI). Reaction of the subunit (Fig. 2B) or E109A complex (Fig. 2C) with L-serine in the absence of CsCl yields an absorption spectrum with maximum absorbance at 424 nm. This intermediate is the external aldimine of pyridoxal phosphate with L-serine (E-Ser in Fig. SI) and exhibits an intense fluorescence emission at 510 nm. Addition of increasing concentrations of CsCl to the subunit (Fig. 2B) or the E109A complex (Fig. 2C) results in decreased absorbance at 424 nm, increased absorbance at 340 nm, and decreased fluorescence emission at 510 nm (fluorescence data not shown). Thus CsCl alters the equilibrium distribution of ES-Ser and ES-AA intermediates with the subunit and the E109A complex.


Figure 2: Effects of CsCl concentration on absorption spectra in the presence or absence of 50 mML-serine. A, wild type complex; B, wild type subunit; C, E109A complex.




Figure SI: Scheme I. Reactions of the wild type tryptophan synthase complex.




DISCUSSION

Globular proteins are flexible molecules and can have two or more conformations in solution. Ligand or substrate binding may produce changes in protein conformation or alter the equilibrium distribution of preexisting conformations. The results reported above show the effects of two ligands (Cs and -glycerol 3-phosphate) on the catalytic properties and spectroscopic properties of wild type and mutant forms of tryptophan synthase. We conclude that Cs stabilizes the active conformation of tryptophan synthase and partially repairs the deleterious effects of a mutation that leads to an inactive conformation.

CsAlters the Catalytic Properties of Tryptophan Synthase

Our analysis (Table 3) of the activity data in Fig. 1indicates that Cs binds to two classes of sites in the wild type and E109A complexes and to one class of sites in the wild type subunit. Low concentrations of Cs activate the wild type complex (K = 25 mM) and the wild type subunit (K = 43 mM). Because 50 mM Na is present in our assay mixture, the activity of the wild type complex in the absence of CsCl is significantly higher than the activity in the absence of any monovalent cation(27, 28, 29, 30, 31) . The cited results indicate that monovalent cations convert the wild type complex from conformation I that is less active in the reaction to a more active conformation II as shown in Table 1. Our results in Table 3indicate that low concentrations of Cs convert the wild type complex from a less active form X to a more active form Y. High concentrations of Cs decrease the activity of the wild type complex. The shape of the curve in Fig. 1A (-GP) suggests that these high concentrations of CsCl may have a solvent effect and favor a less active conformation Z (Table 3). Since 3 M CsCl does not alter the far- and near-UV circular dichroism spectra of the wild type complex (data not shown), effects of this high concentration of CsCl must have subtle, rather than gross, effects on the conformation. Addition of GP, an analogue of the subunit substrates indole-3-glycerol phosphate and D-glyceraldehyde 3-phosphate, inhibits the activity of the wild type complex in the reaction (Fig. 1A) as found previously(39) . Inhibition is greatest near the concentration of CsCl that gives maximum activity in the absence of GP. This result suggests that the most active form of the complex (form Y in Table 3) is most susceptible to this allosteric inhibition. It is possible that the step which is inhibited by -glycerol 3-phosphate is rate-limiting for form Y of the complex but is only partially rate-limiting for forms X and Z.

CsAlters the Spectroscopic Properties of the Subunit

Our finding CsCl alters the equilibrium distribution of enzyme-substrate intermediates (ES-Ser and ES-AA in Fig. SI) with the subunit (Fig. 1B) is consistent with early studies on the effects of NH on the spectroscopic properties of the subunit with L-serine(24) . NH was also found to alter the presteady state kinetics of interconversion of ES-Ser and ES-AA(26) . In some ways high concentrations of NH mimic the effects of the subunit on the properties of the subunit. NH or subunit increases the rate of formation of E-Ser and increases the rate of conversion of E-Ser to E-AA (Fig. SI). The conversion of E-Ser to E-AA is rate-limiting in the absence of NH or subunit(25) .

CsPartially Repairs a Conformational Defect in a Mutant Enzyme

Addition of Cs to the complex having the E109A mutation in the subunit partially restores the activity with L-serine and indole (Fig. 1C) and makes the spectroscopic properties in the presence of L-serine (Fig. 2C) more like those of the wild type complex (Fig. 2A). These results provide evidence that a high concentration of Cs stabilizes conformation II of the E109A complex that is more active in the reaction. Alternatively, high concentrations of Cs might prevent dissociation of the E109A complex to separate and subunits under assay conditions. A strong argument against this possibility is provided by our previous finding that addition of GP results in an 80% inhibition of the activity of the wild type and E109A complexes in the -replacement reaction with -chloro-L-alanine and indole, but does not inhibit the corresponding activity of the separate subunit(17) .

Related previous studies have shown that addition of a high concentration of NH partially restores the activity of a mutationally altered form of the subunit found in E. coli strain A2B17(22) . This type of mutant has been termed ``repairable'' because it has activity with L-serine in the reaction in the presence, but not in the absence, of the subunit. Ammonium ions often work synergistically with subunits in the activation of other repairable subunits(40) . A high concentration of NH also partially restores the activity of a mutant form of the subunit (B8) that has an amino acid substitution in the ``hinge'' region(41, 42) . The results suggest a functional role for the hinge region in the process of conformational switching. Studies in progress in our laboratory reveal that moderate concentrations of Cs or NH also partially restore the activities of a number of other mutant forms of the subunit and complex. Taken together the results of these studies support the model in Table 1that monovalent cations and the subunit stabilize conformation II of the wild type and mutant subunits that is more active in the reaction.

Crystallographic Studies of Cation Binding Sites in Pyridoxal Phosphate Enzymes

It is important to obtain additional evidence for the nature of the conformational change postulated in Table 1. Three other pyridoxal phosphate-dependent enzymes which are activated by monovalent cations (tyrosine phenol-lyase(43, 44) , tryptophanase(44, 45, 46) , and dialkylglycine decarboxylase(47) ) have recently been examined by x-ray crystallography(48, 49, 50, 51, 52) . (For an excellent recent review, see (30) ). Because the available x-ray structures show that the monovalent cation binding sites are not located in the active site, the bound cations should be classified as allosteric effectors(30) . These allosteric effectors may activate the enzymes by stabilizing the active conformation or may play a more dynamic role. For an enzyme such as tryptophan synthase that catalyzes a reaction through a series of steps (see Fig. SI) monovalent cation binding could selectively lower the activation energy for a particular step and thus alter the equilibrium distribution of intermediates and the kinetics of specific steps(30) .

The crystal structure of pyridoxal phosphate-dependent 2,2-dialkylglycine decarboxylase reveals the location of two binding sites for alkali metal ions. One is located near the active site and accounts for the dependence of activity on K or Rb(49, 51, 52, 53) . The exchange of Na for K at the location near the active site results in a gross change in the coordination geometry, in concerted rearrangement of the conformations of Ser and Tyr, and in small structural changes extending far beyond the immediate surroundings of the metal ion(49, 51, 52) . The changes in the conformations of Ser and Tyr, which are in the active site, may disrupt the productive binding of substrate. This change in conformation could drive the enzyme into an inactive (or low activity form) in which the bound substrate and the active site functional groups and/or the pyridoxal phosphate ring are improperly aligned for catalysis(30) . These important studies provide the groundwork for understanding the structural and functional roles of monvalent cations in other pyridoxal phosphate enzymes.

Structure and Function of Tryptophan Synthase

The three-dimensional structure of the tryptophan synthase complex from S. typhimurium has been reported (53) and described in several reviews(3, 5, 30) . Because the active sites of the and subunits are 25 Å apart, the observed effects of ligands that bind to each subunit on the properties of the partner subunit are allosteric in nature. Thus ligands that bind to the active site of the subunit produce allosteric effects on the individual steps in reactions catalyzed by the subunit (Fig. SI). Studies by Peracchi et al.(28, 29) and Dunn (27, 30, 31) and our studies reported herein show that monovalent cations also act as allosteric effectors of these subunit reactions. In addition, Woehl and Dunn (30, 31) find that monovalent cations are essential for the allosteric activation of the site by formation of E-AA (see Fig. SI) at the site. The cited results and results herein support a role for monovalent cations as a switch that converts the low activity conformation I to a high activity conformation II (Table 1). Crystallographic studies of the tryptophan synthase complex in progress show that Na, K, or Cs binds to a site near, but not in, the active site of the subunit.()The results of these studies should give new insights into the roles of monovalent cations in tryptophan synthase.

In conclusion, to explain our finding that the wild type subunit of tryptophan synthase and a mutant form of the complex ( subunit-E109A) have no or low activity in reactions with L-serine but high activity in reactions with -chloro-L-alanine, we propose that that these enzymes exist in a conformation (I) that results in the improper alignment of the weak hydroxyl leaving group of L-serine for -elimination. We suggest that bound Cs stabilizes conformation II in which the hydroxyl group of L-serine is properly aligned for -elimination. Thus Cs may stabilize alternative conformations of the wild type subunit and E109A complex that have structural and catalytic properties more similar to those of the wild type complex.


FOOTNOTES

*
A preliminary report of this work was presented at the 9th Meeting of Vitamin B and Carbonyl Catalysis and the Third Symposium on PQQ and Quinoproteins in Capri, Italy, May 22-27, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: NIH, Bldg. 8, Rm. 2A09, Bethesda, MD 20892-0830. Tel.: 301-496-2763; Fax: 301-402-0240; ewmiles{at}helix.nih.gov

The abbreviations used are: GP, DL--glycerol 3-phosphate; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

D. R. Davies, personal communication.


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