Importance of Conserved and Variable C-terminal Residues for the Activity and Thermal Stability of the beta  Subunit of Tryptophan Synthase*

(Received for publication, October 30, 1996, and in revised form, January 9, 1997)

Li-hong Yang , S. Ashraf Ahmed , Sangkee Rhee § and Edith Wilson Miles

From the  Enzyme Structure and Function Section, Laboratory of Biochemical Pharmacology and the § Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

To assess the functional roles of helix 13 and of the conserved and variable residues in the C-terminal region (residues 378-397) of the tryptophan synthase beta  subunit, we have constructed four C-terminal truncations and 12 point mutations. The effects of these mutations on kinetic and spectroscopic properties and thermal stability are reported here. The mutant beta  subunits all form stable alpha 2beta 2 complexes that have been purified to homogeneity. The mutant alpha 2beta 2 complexes are divided into two classes on the basis of activity in the reaction of L-serine with indole to form tryptophan. Class I enzymes, which have mutations at Arg-379 or Asp-381 or truncations (384-397 or 385-397), exhibit significant activity (1-38% of wild type). Class II enzymes, which have mutations at Lys-382 or Asp-383 or truncations (382-397 or 383-397), exhibit very low activity (<1% of wild type). Although Class II enzymes have drastically reduced activity in the reaction of L-serine with indole and an altered distribution of enzyme-substrate intermediates in the reaction of L-serine with beta -mercaptoethanol, they retain activity in the reaction of beta -chloro-L-alanine with indole. Correlation of the results with the three-dimensional structure of the alpha 2beta 2 complex suggests that Lys-382 and Asp-383 serve important roles in a proposed "open" to "closed" conformational change that occurs in the reactions of L-serine. Because mutant beta  subunits having C-terminal truncations (383-397 or 384-397) undergo much more rapid thermal inactivation at 60 °C than the wild type beta  subunit, the C-terminal helix 13 stabilizes the beta  subunit.


INTRODUCTION

The bacterial tryptophan synthase alpha 2beta 2 complex (EC 4.2.1.20) is a useful model system for probing the relationships between enzyme structure and function and the mechanisms of intersubunit communication (for reviews see Refs. 1-4). The three-dimensional structure of the alpha 2beta 2 complex from Salmonella typhimurium revealed that the four polypeptide chains are arranged in a nearly linear alpha beta beta alpha order (5). The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, termed the N-domain (residues 1-52 and 85-204 shown in yellow in Fig. 1A) and the C-domain (residues 53-84 and 205-397 shown in cyan and red in Fig. 1A). The pyridoxal phosphate coenzyme is located in the interface between these two domains and interacts with residues from both domains. Portions of the two domains possess a high level of structural homology and are nearly superimposable, suggesting that they may have evolved by gene duplication and fusion. The core region of the C-domain terminates at residue 377 and thus excludes the C-terminal residues 378-397. Residues 383-393 form a helix (helix 13) that protrudes from the center of the beta  subunit into solvent. Three residues in helix 13 (Ile-384, His-388, and Leu-391) are involved in beta /beta interaction (Fig. 1B). The side chains of Lys-382 and Glu-350 form a salt bridge located below the plane of the pyridoxal phosphate ring in the active center of the beta subunit (Fig. 1B and Fig. 5 in Ref. 6).


Fig. 1. A, ribbon diagram of the three-dimensional structure of the tryptophan synthase alpha 2beta 2 complex from S. typhimurium (5). The alpha  subunits are in green, the N-domain of the beta  subunit (residues 1-52 and 85-204) is in yellow; the C-domain residues 53-84 and 205-382 are in cyan, and residues 378-393 are in red; C-terminal residues 394-397 are not visible in the structure and are not shown. The pyridoxal phosphate coenzyme is in black. B, stereo ribbon diagram of the central section from A. Structural elements are labeled according to Ref. 5: alpha , alpha -helix; beta , beta  strand. Side chains that are deleted or mutated in this work (see Fig. 2) are shown in lavender. Side chains of Glu-350 and Gln-142 are shown in green.
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Alignment of the sequences of the tryptophan synthase beta  subunit or beta  domain from 24 species (see supplementary material in the electronic appendix to Ref. 7) reveals that residues corresponding to 384-397 of the beta  subunit from S. typhimurium are variable, whereas the preceding residues 378-383 are identical (Fig. 2). Several other pyridoxal phosphatedependent enzymes, including dehydratases and synthases, have also been aligned with the tryptophan synthase beta  subunit and assigned to the beta  family (8) or to Fold type II (7). The sequence similarity of these other enzymes with the beta  subunit is very low in the C-terminal region (beyond Glu-350 in the beta  subunit from S. typhimurium).


Fig. 2. Sequence of C-terminal residues 378-397 of the tryptophan synthase beta  subunit from S. typhimurium and structural elements found in the three-dimensional structure (5) (r, random coil; h, helix; ?, cannot be seen). Multiple alignment of 24 beta  subunit or domain sequences (7) shows that residues corresponding to 378-383 (underlined) of the beta  subunit from S. typhimurium are identical, whereas 384-397 are variable. We have constructed and characterized the four indicated C-terminal truncation mutant proteins and 12 mutant proteins having single amino acid replacements at positions 379, 381, 382, and 383 marked by *; the construction and initial characterization of five mutant beta  subunits altered at position 382 has been reported recently (6).
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To investigate the roles of helix 13 and of the conserved and variable residues in the C-terminal region, we have constructed four C-terminal truncations and 12 point mutations (Fig. 2) using methods based on PCR1 (6, 9). The effects of these mutations on kinetic and spectroscopic properties and thermal stability are reported here. Our results show that residues in the C-terminal region are important for the thermal stability and activity of the alpha 2beta 2 complex but are not essential for catalysis. Alteration or deletion of Lys-382 or Asp-383 drastically reduces activity in the reaction of L-serine with indole and alters the substrate specificity and spectroscopic properties. We correlate these results with data on the three-dimensional structure of the alpha 2beta 2 complex (5, 10)2 and with models depicting ligand-mediated conformational changes of the tryptophan synthase alpha 2beta 2 complex (11-16). Our results suggest that Lys-382 and Asp-383 are involved in an open to closed conformational transition that activates the alpha 2beta 2 complex. Initial aspects of portions of this work have been reported (6, 9).


EXPERIMENTAL PROCEDURES

Enzymes and Chemicals

Enzymes and chemicals used for recombinant DNA methods and DNA sequencing were as before (6). Indole-3-glycerol phosphate was prepared as described (17). beta -Chloro-L-alanine, D-glyceraldehyde-3-phosphate dehydrogenase, and other common chemicals were from Sigma. Buffer B was 50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8. A synthetic peptide (NH2-DIFTVHDILKA-COOH), corresponding to beta  subunit residues 383-393, was custom synthesized by Bio-Synthesis Corp., Lewisville, TX.

Bacterial Strains and Plasmids, Growth of Cells, and Purification of Enzymes

The Escherichia coli host strain CB149 lacks the trp operon (17). Plasmids pEBA-10, pEBA-6, and pEBA-4A8 (6) express the S. typhimurium tryptophan synthase alpha 2beta 2 complex, beta  subunit, and alpha  subunit, respectively. Cultures of the host harboring wild type or mutant plasmids (see below) were grown, and enzymes were induced with IPTG as described (6). Enzymes were purified from the disrupted cells from 100-ml cultures. Purification of the wild type and mutant alpha 2beta 2 complexes (18) utilized crystallization from crude extracts followed by recrystallization. The combined yields of alpha  and beta  subunits were greater than 70% of the total soluble proteins, as reported recently for the five complexes with mutations in beta  subunit residue 382 expressed under the same conditions (6). The amounts of purified alpha 2beta 2 complex obtained from 100-ml cultures ranged from 23 to 90 mg. Each purified alpha 2beta 2 complex gave only two bands on SDS-polyacrylamide gel electrophoresis that correspond to the alpha  and beta  chains (data not shown; see Fig. 3 in Ref. 6 for analogous results) and an absorption spectrum showing the presence of bound pyridoxal phosphate (see below). Our finding that each overexpressed mutant beta  subunit gave a sharp band on SDS-polyacrylamide gel electrophoresis and was obtained in high yield indicates that there was no degradation of any of these overexpressed proteins overall or from the carboxyl end.

The alpha  and beta  subunits were purified as described (19). Although the Delta 385-397 mutant protein was expressed as the beta  subunit by the plasmid pEBA-6 (Delta 385-397) as described below, this beta  subunit was purified as the alpha 2beta 2 complex for three reasons as follows: 1) higher yields of alpha 2beta 2 complex than beta  subunit are obtained by the procedures used; 2) a mutant beta  subunit may be stabilized by interaction with the alpha  subunit; and 3) alpha 2beta 2 complexes were used in the experiments reported. The Delta 385-397 beta  subunit was purified as the alpha 2beta 2 complex after mixing the harvested cells from two 100-ml cultures (E. coli CB 149/pEBA-6 (Delta 385-397) and E. coli CB 149/pEBA-4A8), which express the mutant beta  subunit and the wild type alpha  subunit, respectively.

PCR-based Mutagenesis

The expression vectors pEBA-10 and pEBA-6 were used as templates for quick and convenient mutagenesis by megaprimer PCR (6). Mutagenic primers used in the construction of the missense mutations and C-terminal truncations were as follows, where base changes are underlined and mixed bases at the same position are in parentheses: R379P, R379K, or R379Q, 5'-T CTC TCT GGC <UNL>(CA)(CA)A</UNL> GGA GAT AAA G-3'; D381S, D381N, or D381Y, 5'-AT CTC TC<UNL>C</UNL> GG<UNL>A</UNL> CGC GGA <UNL>(TA)(AC)T</UNL> AAA GAC AT-3' (n.b. TC<UNL>C</UNL> GG<UNL>A</UNL> introduces an AccIII site); Delta 382-397, 5'-C CGC GGA GAT <UNL>TAG</UNL> GAC ATC TTT A-3'; D383A, 5'-GA GAT AAA <UNL>GCC</UNL> ATC TTT ACC G-3'; Delta 383-397, 5'-C GGA GAT AAA <UNL>TAG</UNL> ATC TTT ACC G-3'; Delta 384-397, 5'-A GAT AAA GAC <UNL>TAG</UNL> TTT ACC GTA C-3'; Delta 385-397, 5'-AAA GAC ATC <UNL>TAG</UNL> ACC GTA CAC G-3'. The TC<UNL>TAG</UNL>A in the oligomer for Delta 385-397 introduces an XbaI site. As a result, the mutagenized PCR fragment has two XbaI sites that prevent recloning into pEBA-10. Therefore, the mutagenized fragment was cloned into pEBA-6 after additional manipulation. The fragment was first inserted into the linear pCRII as described (6). The product was cut with EcoRI, and the resulting small fragment was filled in by Klenow and then cut by SphI to yield a fragment that has an SphI site at one end and EcoRI/fill in at the other end. This fragment was then introduced into the SphI and HindIII/fill in sites of pEBA-6. The mutant plasmids are designated as parent (mutation) (e.g. pEBA-6 (Delta 385-397) and pEBA-10 (R379Q)). The construction of five mutant proteins with amino acid replacements at position 382 was as described (6).

Enzyme Assays

One unit of activity is the amount of enzyme that gives rise to formation of 0.1 µmol of product per 20 min at 37 °C. Activities of the alpha 2beta 2 complex in the conversion of indole (0.2 mM) and L-serine or beta -chloro-L-alanine (40 mM) to L-tryptophan were determined by a spectrophotometric assay in the presence of an approximately 3-fold excess of alpha  subunit (16, 20). CsCl (0.5 M) was added in some assays where indicated. The activities of the alpha 2beta 2 complex in the alpha  reaction (conversion of indole-3-glycerol phosphate to indole and D-glyceraldehyde 3-phosphate) and in the alpha beta reaction (conversion of indole-3-glycerol phosphate and L-serine to tryptophan and D-glyceraldehyde 3-phosphate) were measured by a spectrophotometric assay coupled with D-glyceraldehyde-3-phosphate dehydrogenase in the presence of excess alpha  subunit (21).

Spectroscopic Methods

Absorption spectra utilized a Hewlett-Packard 8452 diode array spectrophotometer. Fluorescence measurements were made in a Delta -Scan 1 (Photon Technology International) dual excitation spectrofluorimeter. Fluorescence titrations of enzymes with L-serine measured the increase in fluorescence emission at 510 nm (excitation at 420 nm) upon incubation of the alpha 2beta 2 complex (0.26-0.52 µM alpha beta pair) with 0.01-10 mM L-serine at 25 °C (22-24). Kd values for L-serine were obtained by hyperbolic curve fitting of the change in fluorescence Delta F using Equation 1:
&Dgr;F=&Dgr;F<SUB><UP>max</UP></SUB> ∗ [<UP><SC>l</SC></UP><UP>-serine</UP>]/K<SUB>d</SUB>+[<UP><SC>l</SC></UP><UP>-serine</UP>] (Eq. 1)
Kd values for beta -chloro-L-alanine were obtained from the competitive inhibition by beta -chloro-L-alanine of the fluorescence of the enzyme complexes with L-serine. In Method 1, several concentrations of beta -chloro-L-alanine were used (0, 0.2, 0.5, 1, and 3 mM for the wild type and 0, 10, 20, 40, and 60 mM for the K382N alpha 2beta 2 complex), and Ki values were obtained by linear fit of Kobs/Kd versus [beta -chloro-L-alanine] using Equation 2:
K<SUB><UP>obs</UP></SUB>/K<SUB>d</SUB>=1+[&bgr;-<UP>chloro-<SC>l</SC>-alanine</UP>]/K<SUB>i</SUB> (Eq. 2)
In Method 2, a single concentration of beta -chloro-L-alanine was used (0.5 or 4 mM), and Ki values were obtained from double-reciprocal plots of Delta F versus [L-serine] using Equation 3:
<UP>intercept on</UP> 1/[<UP><SC>l</SC></UP><UP>-serine</UP>] <UP>axis</UP>= (Eq. 3)
<UP>−</UP>1/K<SUB>d</SUB> (1+[&bgr;-<UP>chloro</UP>-<SC>l</SC>-<UP>alanine</UP>]/K<SUB>i</SUB>)

Thermal Inactivation

Enzyme solutions (2 mg/ml alpha 2beta 2 complex in ~10 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 0.1 mM pyridoxal phosphate, 0.1 M potassium phosphate, and 1 mM EDTA at pH 7.8) were incubated in Eppendorf tubes in a water bath at 60 °C for various times. Tubes were chilled to 4 °C for 30 min or more and then centrifuged. Aliquots of supernatant solutions were assayed for enzymatic activity in the beta  reaction with indole and L-serine in the presence of a 3-fold excess of wild type alpha  subunit.


RESULTS

To investigate the functional and structural roles of the C-terminal region of the tryptophan synthase beta  subunit, we have engineered 12 point mutations of the conserved residues Arg-379, Asp-381, Lys-382, and Asp-383 and 4 C-terminal truncations. We did not investigate the effects of mutations altering the conserved residues Gly-378 and Gly-380 in this work because these residues do not interact with other residues and have not been implicated previously as important residues. Initial studies on five mutant proteins altered at position 382 have been reported (6). The engineered mutant beta  subunits were all expressed in high yield and purified as stable alpha 2beta 2 complexes (see "Experimental Procedures").

Evidence for Structural Integrity of the Mutant alpha 2beta 2 Complexes: Activation of the alpha  Subunit

Interaction of the wild type alpha  and beta  subunits stimulates the catalytic activity of the alpha  subunit in the alpha  reaction (Equation 4) about 50-fold (see Ref. 1 and footnote to Table I).
&agr; <UP>Reaction</UP>: <UP>indole</UP> 3-<UP>glycerol phosphate</UP> ↔ <UP>indole</UP>+<UP><SC>d</SC></UP><UP>-glyceraldehyde</UP> 3-<UP>phosphate</UP> (Eq. 4)
Activities in the alpha  reaction of alpha 2beta 2 complexes containing mutant beta  subunits and wild type alpha  subunit range from 15 to 91% that of the wild type alpha 2beta 2 complex (Table I). Thus, the mutant beta  subunits all stimulate the catalytic activity of the alpha  subunit. This result provides partial evidence for the structural integrity of the mutant alpha 2beta 2 complexes.

Table I.

Specific activities of wild type and mutant alpha 2beta 2 complexes

Specific activities in the alpha , beta , and alpha beta reactions were determined as described under "Experimental Procedures" in the presence of a 3-fold excess of wild type alpha  subunit. The activity of the alpha  subunit alone in the alpha  reaction was 0.6 units/mg or 1-2% that of the wild type alpha 2beta 2 complex. IGP, indole-3-glycerol phosphate; G3P, D-glyceraldehyde 3-phosphate; WT, wild type; Ind, indole; Rxn, reaction.
Enzyme alpha 2beta 2  alpha Rxn (IGP right-arrow G3P)  alpha beta Rxn (Ser + IGP right-arrow G3P) Ratio of alpha beta Rxn/ alpha  Rxn  beta Rxn (Ser + Ind right-arrow Trp) Mutant class

units/mg % units/mg % units/mg %
WT 41 (100) 802 (100) 20 1100 (100)
R379Q 23 57 350 44 15 414 38 I
R379K 28 68 251 31 10 284 26 I
R379P 6 15 26 3 4 14 1 I
D381N 26 64 56 7 2.2 35 3 I
D381S 37 91 187 23 5.1 160 14 I
D381Y 31 78 247 31 8.0 275 25 I
K382N 29 73 13 2 0.4 0.5 0.05 II
K382G 29 72 20 3 0.7 4 0.4 II
K382R 23 58 13 2 0.6 1.6 0.16 II
K382E 27 68 13 2 0.5 0.2 0.02 II
K382P 25 64 24 3 1 3.5 0.4 II
D383A 30 69 14 2 0.5 0.1 0.01 II
 Delta 382-397 14 35 14 2 1 4 0.4 II
 Delta 383-397 27 68 13 2 0.5 2 0.2 II
 Delta 384-397 13 33 66 8 5 86 8 I
 Delta 385-397 15 38 163 20 11 167 15 I

Activity Measurements Distinguish Two Classes of Mutant beta  Subunits

Table I gives the specific activities of the wild type and mutant alpha 2beta 2 complexes in the synthesis of L-tryptophan from L-serine and indole (beta  reaction, Equation 5) and from L-serine and indole-3-glycerol phosphate (alpha beta reaction, Equation 6).
&bgr; <UP>Reaction</UP>: <UP><SC>l</SC></UP><UP>-serine</UP>+<UP>indole</UP>→<UP><SC>l</SC></UP><UP>-tryptophan</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP> (Eq. 5)
&agr;&bgr; <UP>Reaction</UP>: <UP><SC>l</SC></UP><UP>-serine</UP>+<UP>indole</UP>-3-<UP>glycerol phosphate</UP>→<UP><SC>l</SC></UP><UP>-tryptophan</UP>+<UP><SC>d</SC></UP><UP>-glyceraldehyde</UP> 3-<UP>phosphate</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP> (Eq. 6)
The alpha beta reaction is essentially the sum of the alpha  and beta  reactions and requires coupled catalysis by the alpha  and beta  subunits. Reaction of L-serine at the beta  site in the alpha 2beta 2 complex stimulates the rate of indole-3-glycerol phosphate cleavage approximately 20-fold as shown by the ratio of alpha beta reaction:alpha reaction in Table I.

We have arbitrarily classified the mutant alpha 2beta 2 complexes on the basis of the effects of the mutations on the activity in the beta  reaction, the reaction of L-serine with indole. "Class I" enzymes, which have mutations at positions 379 and 381 or truncations after residue 383 (384-397 or 385-397), exhibit significant activity (1-38% of wild type). "Class II" enzymes, which have mutations at positions 382 or 383 or truncations including residues 382 or 383 (382-397 or 383-397), exhibit very low activity (<1% of wild type). The absence of L-serine stimulation of indole-3-glycerol phosphate cleavage by Class II enzymes (Ratio alpha beta reaction/alpha reaction = ~1 in Table I) can be attributed to the lack reaction of L-serine at the beta  site.

Some mutant forms of tryptophan synthase have considerably higher activity in the overall alpha beta reaction than in the individual beta  reaction (25, 26). These results were partly due to a reduced association of the mutant alpha  subunit with the beta  subunit; addition of indole-3-glycerol phosphate during catalysis of the beta  reaction or alpha beta reaction increased association and activity. The finding that the activity of each mutant protein in Class I is quite similar in the alpha beta and beta  reactions (Table I) shows that Class I mutations have no effects on association between the alpha  and beta  subunits that can be detected by these assays of enzymatic activity.

Class II Mutant alpha 2beta 2 Complexes Have Altered Substrate Specificity

Table II compares the activities of the wild type and mutant enzymes in the reaction of beta -chloro-L-alanine with indole to activities in the reaction of L-serine with indole. It is striking that all of the mutant enzymes, except the Class I enzymes with mutations at position 379, have rather high activities with beta -chloro-L-alanine (42-140% of the wild type enzyme). Thus, the Class II mutations, which lead to loss of activity with L-serine and indole, do not prevent the analogous reaction with beta -chloro-L-alanine. Consequently, the substrate specificity ratio, which is defined as the ratio of activity with beta -chloro-L-alanine and indole to activity with L-serine and indole, is much higher for the Class II enzymes (25-500) than for the wild type alpha 2beta 2 complex (0.24) or the Class I mutant proteins (0.11 to 8.7).

Table II.

Substrate specificities and spectroscopic properties of wild type and mutant alpha 2beta 2 complexes

Specific activities in the conversion of indole and either L-serine or beta -chloro-L-alanine to tryptophan were determined as described under "Experimental Procedures" and expressed as percent of activity of the wild type alpha 2beta 2 complex with L-serine in the absence of CsCl (1100 units/mg, see Table I) or in the presence of 0.5 M CsCl (880 units/mg, see Ref. 6). beta  Rxn, beta  reaction, beta -ME, beta -mercaptoethanol, WT, wild type. Values of lambda max were obtained from absorption spectra recorded as described in Fig. 3.
Enzyme alpha 2beta 2  beta Rxn (Ser right-arrow Trp) +CsCla Cl-Ala right-arrow Trp Ratio, Cl-Ala/Ser Class +Ser (lambda max) + CsCl +Ser, beta -ME (lambda max) + CsCl

% % nm nm
WT (100) (100) 24 0.24 350 468
R379Q 38 39 6.2 0.11 I 350 468
R379K 26 29 4.3 0.24 I 350 468
R379P 1 16 2.5 2.5 I 350 /424 468 /495
D381N 3 40 26 8.7 I 350 /424 468
D381S 14 70 33 2.4 I 350 468
D381Y 25 40 14 0.56 I 350 468
K382N 0.05 1.6b 18 360 II 424 424
K382G 0.4 2.2b 18 45 II 424 424
K382R 0.2 2.5b 16 80 II 424 424
K382E 0.02 0.6b 10 500 II 424 424
K382P 0.4 1.1b 19 48 II 424 424
D383A 0.13 10 19 150 II 424 424
 Delta 382-397 0.4 5.2 10 25 II 350 /424 424
 Delta 383-397 0.2 2.4 32 160 II 424 424
 Delta 384-397 8 19 11 1.4 I 350 468
 Delta 385-397 23 108 26 1.7 I 350 468
WT beta 2c 46d 28 14 424e f

a CsCl (0.5 M) was added.
b Data from Ref. 6.
c Data from Ref. 16.
d Data from Ref. 11.
e Data from Ref. 35.
f Not determined.

Effects of Mutations on Binding Constants of L-Serine and beta -Chloro-L-alanine

Table III compares the binding constants of L-serine and beta -chloro-L-alanine for the wild type and several mutant alpha 2beta 2 complexes. The results were obtained by determining the effect of L-serine concentration on the change in fluorescence emission at 510 nm in the presence or absence of fixed concentrations of beta -chloro-L-alanine, a competitive inhibitor that does not form a fluorescent external aldimine (22-24). The Kd values of L-serine and beta -chloro-L-alanine are similar for all of the enzymes except K382N. Although the binding constant of L-serine (3 mM) for the K382N alpha 2beta 2 complex is markedly elevated, this enzyme should be saturated by the concentration of L-serine (40 mM) used in the assay. The K382N alpha 2beta 2 complex exhibits high activity with beta -chloro-L-alanine and indole even though the Kd for beta -chloro-L-alanine (78 mM) is higher than the concentration of beta -chloro-L-alanine (40 mM) used in the assay. Thus, the effects of the Class II mutations on substrate specificity are not attributable to changes in the binding of L-serine or of beta -chloro-L-alanine.

Table III.

Dissociation constants of L-serine and beta -chloro-L-alanine for the wild type (WT) and representative mutant alpha 2beta 2 complexes

Kd values for L-serine and for beta -chloro-L-alanine were determined by fluorescence titrations as described under "Experimental Procedures."
Enzyme alpha 2beta 2 Mutant class L-Serine, Kd  beta -Chloro-L-alanine
Kd Method

mM mM
WT 0.36  ± 0.05 0.95  ± 0.06 1
R379Q I 0.32  ± 0.02 0.47 2
K382N II 2.9  ± 0.1 78  ± 6 1
D383A II 0.21  ± 0.01 0.73 2
 Delta 383-397 II 0.29  ± 0.04 1.2 2

CsCl Partially Repairs Activities of the Mutant alpha 2beta 2 Complexes

Because CsCl activates certain mutant alpha 2beta 2 complexes (11), including those having single amino acid replacements of beta  subunit Lys-382 (6), we have also determined activity in the beta  reaction in the presence of 0.5 M CsCl (Table II). CsCl activates all of the mutant alpha 2beta 2 complexes that have low activity but has little effect on the R379Q and R379K alpha 2beta 2 complexes that have relatively high activities. CsCl results in a striking 16-fold activation of the R379P alpha 2beta 2 complex, which has a much lower activity than R379Q and R379K under standard conditions. CsCl also activates the wild type beta  subunit (11).

Spectroscopic Properties of the Mutant alpha 2beta 2 Complexes

The wild type alpha 2beta 2 complex and beta  subunit catalyze a beta -replacement reaction of L-serine with beta -mercaptoethanol that proceeds through a series of pyridoxal phosphate-substrate intermediates that have characteristic absorption spectra (see Fig. 3 for structures and abbreviations) (27, 28). Whereas an equilibrium mixture of these intermediates accumulates with the wild type alpha 2beta 2 complex, the E-Ser intermediate predominates with the wild type beta  subunit and most of the mutant alpha 2beta 2 complexes (data not shown). Conversion of E-Ser to E-AA is rate-limiting with the wild type beta  subunit (29). Because Cs+ and NH4+ alter the rate of this conversion by the beta  subunit (29) and alter the rates and spectroscopic properties of some mutant enzymes (11), we have determined the absorption spectra obtained upon reaction of L-serine and of L-serine and beta -mercaptoethanol in the presence of 0.5 M CsCl (Fig. 3 and Table II). Fig. 3, A and B, shows the spectra obtained in the presence of 0.5 M CsCl with the R379Q alpha 2beta 2 complex (a representative of Class I) and with the Delta 383-397 alpha 2beta 2 complex (a representative of Class II), respectively. The three spectra obtained with the R379Q alpha 2beta 2 complex are essentially identical to those of the wild type alpha 2beta 2 complex under the same conditions. The enzyme alone exhibits a major peak centered at 410 nm due to the internal aldimine (E). Reaction with L-serine yields a complex spectrum with a major peak centered at 340-350 nm, which is ascribed to the external aldimine between pyridoxal phosphate and aminoacrylate (E-AA). Reaction of L-serine and beta -mercaptoethanol yields a major peak at 468 nm, which is ascribed to a quinonoid (E-Q) intermediate formed upon addition of beta -mercaptoethanol to the aminoacrylate intermediate. The Delta 383-397 enzyme alone also exhibits a peak at 410 nm (E). Addition of L-serine yields a major peak at 424 nm which is ascribed to the external aldimine between pyridoxal phosphate and L-serine (E-Ser). The spectrum is unchanged by the further addition of beta -mercaptoethanol.


Fig. 3. Effects of ligands on the absorption spectra of mutant alpha 2beta 2 complexes. Top, reaction of the enzyme (E) with L-serine to form the external aldimine with L-serine (E-Ser) which is dehydrated to the aldimine of aminoacrylate (E-AA). Addition of beta -mercaptoethanol (beta ME) yields a quinonoid intermediate (E-Q) which is then protonated to yield the aldimine of S-hydroxyethylcysteine (E-S-HEC) followed by release of product (S-HEC) to regenerate E. The absorption maximum is shown beneath each intermediate. Corresponding structures are shown on the figure near the corresponding absorption bands. The designation of intermediates as "open" or "closed" is based on experiments using 8-anilino-1-naphthalenesulfonate as a conformational probe (13). Absorption spectra were recorded on solutions of enzymes (1 mg/ml in Buffer B containing 0.5 M CsCl) before (1) or after (2) addition of a 0.05 volume of 1 M L-serine and after further addition (3) of a 0.05 volume of 1 M beta -mercaptoethanol. A, R379Q (representative of Class I), B, Delta 383-397 (representative of Class II).
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Table II presents the wavelength of maximum absorbance in the presence of L-serine or of L-serine plus beta -mercaptoethanol for the wild type alpha 2beta 2 complex and beta  subunit and for each mutant alpha 2beta 2 complex in the presence of CsCl. In general, the three spectra for each mutant alpha 2beta 2 complex in Class I were similar to those of the wild type alpha 2beta 2 complex, whereas the spectra for each mutant alpha 2beta 2 complex in Class II were similar to those of the Delta 383-397 alpha 2beta 2 complex and of the wild type beta  subunit. Thus, CsCl partially restores the wild type spectral properties of the Class I mutant alpha 2beta 2 complexes but not of the Class II mutant alpha 2beta 2 complexes.

Attempted Peptide Rescue of the C-terminal Truncation Mutant Delta 383-397

Addition of a synthetic peptide corresponding to residues 1-9 of the tryptophan synthase beta  subunit partially restores the activity of a mutant beta  subunit having residues 1-9 deleted (30). To test whether a peptide corresponding to the deleted helix 13 could restore the activity and spectral properties of the mutant beta  subunit having residues 383-397 deleted, we mixed the Delta 383-397 alpha 2beta 2 complex (7.8 µM) with the peptide corresponding to residues 383-393 (1.05 mM) in Buffer B in the absence and presence of 0.5 M CsCl at 25 °C for 90 min. Assays of the activity with L-serine and indole in the presence of CsCl (as described in Table II in the absence of peptide) showed that addition of the peptide had no effect on the activity. Similarly, the peptide did not affect the absorption spectra of this mutant enzyme and its enzyme-substrate complexes either in the absence of CsCl or in the presence of CsCl (as described in Fig. 3B in the absence of peptide).

C-terminal Truncation Mutant alpha 2beta 2 Complexes Are Thermolabile

Fig. 4 shows the rates of thermal inactivation of the wild type and three mutant beta  subunits upon incubation of the corresponding alpha 2beta 2 complexes at 60 °C. Because the alpha  subunits in the holo-alpha 2beta 2 complexes undergo thermal inactivation at 60 °C (31), the activities of the beta  subunits were determined in the presence of added excess alpha  subunit. The results show that the beta  subunits in the two C-terminal truncation mutant alpha 2beta 2 complexes examined (Delta 383-397 and Delta 384-397) were much more thermolabile than the wild type beta  subunit. The D381Y beta  subunit was somewhat more thermolabile than the wild type beta  subunit.


Fig. 4. Rates of thermal inactivation of wild type and mutant beta  subunits in the alpha 2beta 2 complex at 60 °C. Thermal inactivation and enzyme assays were as described under "Experimental Procedures" for wild type (open circle ), D381Y (triangle ), Delta 383-397 (black-square), and Delta 384-397 (bullet ).
[View Larger Version of this Image (20K GIF file)]



DISCUSSION

The experiments described above were aimed at assessing the functional roles of the conserved and variable residues and of helix 13 in the C-terminal region of the beta  subunit. Because the C-terminal residues 378-397 are outside of the core region of the C-terminal domain (5) and are not conserved in related enzymes (7), these residues may have evolved to have special functions, such as stabilizing the beta 2 dimer.

The four beta  subunit C-terminal truncations and 12 point mutations were all expressed in high yield as stable alpha 2beta 2 complexes ("Experimental Procedures" and Ref. 6), contained bound pyridoxal phosphate that forms enzyme-substrate intermediates (Fig. 3 and Table II), and activated indole-3-glycerol phosphate cleavage by the alpha  subunit (Table I). Thus, the residues deleted (Delta 382-397) or altered (379, 381, 382 and 383) are not required for overall folding of the beta  subunit, for association of the beta  subunit with the alpha  subunit to form an alpha 2beta 2 complex, or for activation of the alpha  subunit. These results document the structural integrity of these mutant beta  subunits. The beta  subunit C-terminal helix 13 may serve a structural role because the mutant proteins having residues 383-397 or 384-397 deleted were much more thermolabile (Fig. 4).

Residues 379 and 381-397 are not essential for catalysis because all of the mutant enzymes exhibit some activity in the reaction of L-serine with indole (0.01-38% of wild type) and quite significant activity in the reaction of beta -chloro-L-alanine with indole (10-140% of wild type) (Table II). We have divided the mutant proteins into two classes, I and II, based on activity in the reaction of L-serine with indole. Class I mutant proteins are more similar to the wild type enzyme than Class II mutant proteins in activity with L-serine and indole, in substrate specificity, and in spectroscopic properties (Tables I and II and Fig. 3). The results with Class I mutant proteins show that Arg-379, Asp-381, and residues 384-397 are not very important for catalytic activity. Although substitution of Arg-379 by Lys or Gln has minor effects on the catalytic properties, substitution by Pro (R379P) has more drastic effects. This result is consistent with the finding that E. coli cells having this mutation in the trpB gene are unable to convert indole to tryptophan (32). Our results do not explain why the R379P mutant cells were found to produce more indole than cells with a trpB mutation at position 382 (32). The activity in the alpha  reaction is in fact lower than that of any of the other mutant proteins. The proline replacement may alter the backbone geometry and have a more deleterious effect on the conformation of the beta  subunit than the more conservative Lys and Gln replacements.

Class II mutant proteins have extremely low activities in the reaction with L-serine and indole but have activities close to that of wild type in the reaction of beta -chloro-L-alanine with indole (Tables I and II). These mutant proteins all have a substitution or deletion of Lys-382 or of Asp-383. These results support and extend observations that the mutation of Lys-382 results in inactivation (1, 32, 33). The D383A and Delta 383-397 mutant proteins lack the conserved residue Asp-383. The finding that these mutant proteins have extremely low activity in the reaction of L-serine and indole provides the first evidence that Asp-383 is very important for the catalysis of this reaction. The effects of the Class II mutations on activity in the reaction of L-serine with indole are not attributable to changes in the binding of L-serine because the Kd values of L-serine for the mutant enzymes (Table III) are all lower than the concentration of L-serine (40 mM) used in the assay. The importance of Lys-382 for tight binding of L-serine and beta -chloro-L-alanine may be attributable to formation of a salt bridge between Lys-382 and Glu-350 (see Fig. 1B and Ref. 6). This salt bridge could stabilize the substrate binding site. An additional role for Lys-382 in a substrate-induced conformational change is discussed below.

Relation of Class II Mutant Proteins to a Model for Ligand-mediated Conformational Changes

Class II mutant proteins exhibit altered substrate specificity and an altered distribution of enzyme-substrate intermediates in the reaction of L-serine and beta -mercaptoethanol (Table II and Fig. 3). These results are now discussed in relation to previous studies of the conformational states of tryptophan synthase. Dunn and co-workers (12-15) have drawn upon data from a number of investigations of tryptophan synthase by different groups of investigators to formulate a model depicting ligand-mediated conformational changes that occur during the course of the alpha beta reaction. The model postulates that the alpha  and beta  subunits undergo transitions between open and closed conformations which function to coordinate the catalytic activities and promote diffusion of the indole intermediate. Recent investigations using 8-anilino-1-naphthalenesulfonate binding as a probe have identified three distinct conformations of the alpha 2beta 2 complex associated with different liganded states of the alpha  and beta  subunits (13). The conformation of the beta  subunit stabilized by E-AA and E-Q has been designated as closed, whereas the conformation stabilized by E-Ser and E-S-hydroxyethylcysteine has been designated as open (see diagram at the top of Fig. 3) (13).

In the reaction of L-serine with beta -mercaptoethanol, the E-AA (closed) intermediate predominates in the wild type and Class I mutant enzymes, whereas the E-Ser (open) intermediate predominates in the Class II mutant enzymes (Table II and Fig. 3). This result indicates that the mutations hinder the ligand-mediated transition from an open to a closed structure either by stabilizing the open structure or by destabilizing the closed structure. The E-Ser intermediate also predominates in several other alpha 2beta 2 complexes having mutations in the beta  subunit (6, 11, 16). We have postulated that the wild type beta  subunit and these mutant alpha 2beta 2 complexes have a conformation (termed "open") that results in a poor alignment of the weak hydroxyl leaving group of L-serine for protonation and beta -elimination. These enzymes may have much higher activity with beta -chloro-L-alanine because this substrate has a strong leaving group that does not require protonation. The finding that addition of CsCl (0.5 M) activates a number of open mutant proteins and the wild type beta  subunit suggests that Cs+ may stabilize a more active alternative conformation of these enzymes (6, 11). CsCl also increases the very low activities of the D383A, Delta 382-397, and Delta 383-397 mutant proteins 10-80-fold (Table II). CsCl may stabilize the more active, closed conformation of the beta  subunit and partially shift the equilibrium from the open to the closed form (11).

Relation of the Mutation Studies to Crystallographic Results

Fig. 1, A and B, shows the locations of residues that have been altered or deleted in the present investigation in the three-dimensional structure of the wild type alpha 2beta 2 complex. The region containing altered residues (residues 378-393) is shown in red. The C-terminal residues 394-397 are not clearly visible in the structure and are not shown. The C-terminal helix 13 from each beta  subunit is located near the beta /beta interaction site and protrudes from the center of the beta  subunit into solvent. Three residues in helix 13 are involved in beta /beta interactions (Fig. 1B). These interactions are Ile-384 to Pro-144, His-388 to Phe-147, and Leu-391 to Phe-147. Two of the conserved residues mutated (Arg-379 and Asp-381) also make beta /beta interactions: the two Arg-379 residues are stacked with each other; Asp-381 interacts with Arg-148. Lys-382 forms an internal salt bridge with Glu-350 of the same beta  subunit. Although the four C-terminal truncations remove the three beta /beta interactions made by Ile-384, His-388, and Leu-391, the mutant beta  subunits all form stable alpha 2beta 2 complexes. Thus, disruption of interactions between six pairs of residues in the alpha 2beta 2 complex is not sufficient to cause disruption of the beta /beta interface. Similarly, single amino acid substitution of Arg-379 or Asp-381 does not cause disruption of the beta /beta interface.

How are ligand-mediated conformational changes in tryptophan synthase related to structural changes? Crystallographic investigations are only beginning to reveal structural effects of ligands. Recent studies reveal that exchange of K+ or Cs+ for Na+ induces local and long range changes in the three-dimensional structure of the tryptophan synthase alpha 2beta 2 complex (10). Studies of a mutant form of the enzyme (beta K87T) having an external aldimine of L-serine at the beta  site and indole-3-propanol phosphate or DL-alpha -glycerol 3-phosphate at the alpha  site show conformational changes that may be related to formation of the closed conformation in solution.2 Conformational changes observed in these structures of the residues investigated here include movements of Arg-141 toward Thr-386 and of Gln-142 toward Lys-382 and Asp-383. The possible involvement of these interactions in the closed conformation may explain why the mutation or deletion of Asp-383 or of Lys-382 in Class II mutant enzymes appears to destabilize the closed form of the enzyme and prevent the open to closed transition.

Because proteins are stabilized by a large network of interactions, mutations are quite likely to interfere with this complex network and destabilize the protein or alter the conformation of the enzyme needed for optimal activity (34). Some proteins are stabilized by better attachment of the N and C termini to the rest of the molecule to prevent "fraying" (34). Deletion of one of these termini may remove these stabilizing interactions and promote fraying. Although the C-terminal helix 13 of the beta subunit is partially exposed to solvent, it does make several interactions in the beta /beta interface. The removal of helix 13 may destabilize the beta  subunit to heat and loosen beta /beta interaction by removing these interactions as discussed above. Addition of a synthetic peptide corresponding to residues 383-393 did not restore the catalytic activity or the spectroscopic properties of the mutant beta  subunit having residues 383-397 deleted.

Conclusions

Our results show that helix 13 is important for thermal stability and that residues 379, 381, 382, and 383 in the C terminus of the beta  subunit are important, but not essential, for catalytic activity. Lys-382 and Asp-383 appear especially important for stabilization of the closed conformation of the enzyme that has optimal activity with L-serine.


FOOTNOTES

*   A preliminary report of portions of this work was presented at the American Society for Biochemistry and Molecular Biology Joint Meeting with the American Association of Immunologists and the American Society for Investigative Pathology, June 1-6, 1996 in New Orleans, LA.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.
   To whom correspondence and reprint requests should be addressed: NIH, Bldg. 8, Rm. 2A09, Bethesda, MD 20892. Tel.: 301-402-1495; Fax: 301-402-0240; E-mail: ewmiles{at}helix.nih.gov.
1   The abbreviations used are: PCR, polymerase chain reaction; E-Ser, external aldimine of L-serine; E-AA, external aldimine of aminoacrylate; E-Q, quinonoid intermediate formed by addition of beta -mercaptoethanol to E-AA.
2   S. Rhee, K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies, submitted for publication.

Acknowledgments

We express our appreciation to Drs. Charles Yanofsky and Peter McPhie for many thoughtful comments and helpful suggestions. We thank Dr. David R. Davies for crystallographic facilities and encouragement. We thank Dr. Roger S. Rowlett for help with data analysis.


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