©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Assembly in the Tryptophan Synthase Complex
STABILIZATION BY PYRIDOXAL PHOSPHATE ALDIMINE INTERMEDIATES (*)

(Received for publication, December 19, 1994)

Utpal Banik S. Ashraf Ahmed Peter McPhie Edith Wilson Miles (§)

From the Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

This work is aimed at understanding subunit assembly in the tryptophan synthase alpha(2)beta(2) complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the beta(2) subunit of tryptophan synthase for subunit association. We utilize a mutant form of the beta(2) subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T alpha(2)beta(2) complex is inactive in reactions catalyzed by the beta(2) subunit but retains activity in the reaction catalyzed by the alpha subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T beta(2) subunit and alpha(2)beta(2) complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit dissociates more readily from the K87T beta(2) subunit than from the wild type beta(2) subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T beta(2) subunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T beta(2) subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type beta(2) subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type beta(2) subunit and by the external aldimine with L-serine in the K87T beta(2) subunit may further stabilize interaction with the alpha subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta(2) subunit.


INTRODUCTION

The bacterial tryptophan synthase alpha(2)beta(2) complex (EC 4.2.1.20) is a useful system for investigating protein-protein interaction and the mechanism of subunit assembly (for reviews see (1, 2, 3, 4, 5, 6) ). The alpha(2)beta(2) complex dissociates into two alpha subunits and one beta(2) subunit. The monomeric alpha subunit catalyzes the alpha reaction. The dimeric beta(2) subunit contains one pyridoxal phosphate at each active site and catalyzes the pyridoxal phosphate-dependent beta reaction. The alpha(2)beta(2) complex catalyzes the alpha and beta reactions and the overall alphabeta reaction, which is formally the sum of the alpha and beta reactions.

The three-dimensional structure of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium(7) reveals that the alpha and beta(2) subunits are arranged in a nearly linear alphabetabetaalpha order. The active sites of the alpha and beta subunits are 25 Å apart and are connected by a tunnel that passes through the alpha/beta interaction site and between the two structural domains of the beta(2) subunit. The pyridoxal phosphate coenzyme is located at the interface between the two structural domains (N-domain and C-domain) of the beta(2) subunit at one end of the tunnel and interacts with residues from both domains. The carbonyl group of pyridoxal phosphate forms an internal aldimine with Lys-87 in the N-domain. The phosphate group of the coenzyme forms hydrogen bonds with Gly-232, Gly-233, Gly-234, Ser-235, Asn-236, and Ala-237 in the C-domain. The pyridine ring nitrogen atom, N1, is close to the sulfhydryl of Cys-230 and within hydrogen bonding distance of the hydroxyl of Ser-377, both residues in the C-domain.

Early studies of the association of the alpha and beta(2) subunits of tryptophan synthase using sucrose gradient centrifugation showed that pyridoxal phosphate partially stabilized the alpha(2)beta(2) complex(8) . The apo-alpha(2)beta(2) complex (minus pyridoxal phosphate) was totally dissociated, the holo-alpha(2)beta(2) complex (plus pyridoxal phosphate) was partially dissociated, and the holo- alpha(2)beta(2) complex in the presence of L-serine was stable. Pyridoxal phosphate and L-serine also increased the apparent subunit association constants measured under assay conditions(8) . The reaction of L-serine with the alpha(2)beta(2) complex resulted in a 400-fold increase in affinity of the beta(2) subunit for the alpha subunit(9) .

In the present work we ask whether binding pyridoxal phosphate per se increases subunit affinity or whether formation of the internal aldimine with beta(2) subunit lysine 87 is required. To answer this question, we use a mutant form of the beta(2) subunit having the lysine 87 replaced by threonine (K87T)(10) . Studies with the K87T alpha(2)beta(2) complex demonstrated that lysine 87 serves critical roles in transimination, catalysis, and product release(11) . The K87T alpha(2)beta(2) complex is inactive in the beta reaction but retains activity in the alpha reaction. The mutant enzyme binds pyridoxal phosphate as the free aldehyde and forms Schiff base intermediates (external aldimines) with L-serine (K87T-Ser) or L-tryptophan (K87T-Trp) at the beta site. Crystallographic analyses of the external aldimines formed by the K87T alpha(2)beta(2) complex with L-serine and L-tryptophan have been carried out at 2Å resolution (^1)and have localized the substrate and product binding sites in the beta(2) subunit(12) . The crystallographic results show that L-serine and L-tryptophan are bound between the N- and C-domains and interact with some residues of the N-domain. The results presented here provide evidence that formation of interdomain bridges by pyridoxal phosphate-aldimine intermediates stabilizes subunit association and stabilizes pyridoxal phosphate binding.


EXPERIMENTAL PROCEDURES

Chemicals and Buffers

Indole-3-glycerol phosphate was synthesized enzymatically and isolated by ion exchange chromatography on DEAE-Sephadex A25 as described(13) . Pyridoxal phosphate, L-serine, NAD, and glyceraldehyde-3-phosphate dehydrogenase were from Sigma. All experiments utilized Buffer B (50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8). Phenylhydrazine hydrochloride was from Eastman.

Enzymes

Wild type (10) and K87T (11) forms of the tryptophan synthase alpha(2)beta(2) complex and wild type alpha and holo-beta(2) subunits (14) from S. typhimurium were isolated and purified to homogeneity. The apo-beta(2) subunit was prepared by treatment of the holo-beta(2) subunit with 10 mM hydroxylammonium chloride(15) . The K87T beta(2) subunit was prepared from K87T alpha(2)beta(2) complex by heat denaturation of the alpha subunit as described for the wild type alpha(2)beta(2) complex(16, 17) . The reduced beta(2) subunit and alpha(2)beta(2) complex were obtained by treating the wild type beta(2) subunit and alpha(2)beta(2) complex with 10 mM sodium borohydride for 30 min followed by dialysis. The K87T alpha(2)beta(2) complex and beta(2) subunit as isolated contain some tightly bound L-serine (about 0.03 and 0.1 mol/mol of beta protomer, respectively)(11) . Schiff base derivatives of the K87T alpha(2)beta(2) complex or beta(2) subunit with L-serine (K87T-Ser) were prepared by incubating the mutant enzyme for 24 h at 23 °C with 40 mML-serine (11) followed by gel filtration on a PD-10 column (Pharmacia Biotech Inc.) in Buffer B. Protein concentrations were determined from the specific absorbance at 278 nm of the alpha(2)beta(2) complex (E = 6.0), the holo-beta(2) subunit (E = 6.5), the apo-beta(2) subunit (E = 5.8), or of the alpha subunit (E = 4.4)(17) . Protein concentrations of the K87T-Ser complexes were determined by the BCA protein assay reagent (Pierce) using purified beta(2) subunit or alpha(2)beta(2) complex as standard.

Spectroscopic and Analytical Methods

Absorption spectra were made using a Hewlett-Packard 8452 diode array spectrophotometer. Time course measurements at single wavelengths were made using a Cary 118 spectrophotometer. Gel filtration experiments were performed using a prepacked Superose HR12 10/30 column (Pharmacia) on a LCC-501 Plus FPLC system (Pharmacia) at a flow rate of 0.5 ml/min at 23 °C. Pyridoxal phosphate content was determined using a phenylhydrazine reagent (2% phenylhydrazine hydrochloride (w/v) in 10 N H(2)SO(4))(18) . SDS-gel electrophoresis was carried out on the Phastgel system (Pharmacia) using 10-15% gradient gels.

Enzyme Assays and Subunit Interchange Experiments

One unit of activity in any reaction is the formation of 0.1 µmol of product in 20 min at 37 °C. Activity in the beta reaction was measured by a direct spectrophotometric assay(17) . The activities of the alpha(2)beta(2) complex in the alpha reaction were measured by spectrophotometric assays coupled with D-glyceraldehyde-3-phosphate dehydrogenase(19) . The kinetics of dissociation of the alpha(2)beta(2) complex was determined by subunit interchange experiments that utilize a two-step system(9) . The alpha(2)beta(2) complex (70 µM beta protomer in Buffer B) was mixed with 20 volumes of beta(2) subunit (70 µM beta protomer in Buffer B) at 25 °C. Aliquots (0.05 ml) were removed at various time intervals and added to 0.95 ml of enzyme assay mixture at 37 °C. When inactive (reduced or K87T) alpha(2)beta(2) complex was mixed with active beta(2) subunit, increase in activity in the alphabeta reaction was determined. When active wild type alpha(2)beta(2) complex was mixed with inactive (reduced or K87T) beta(2) subunit, decrease in activity in the beta reaction was determined. In some cases, 40 mML-serine was premixed with one or both enzymes.


RESULTS

Stability of Enzyme-bound Pyridoxal Phosphate to Dialysis

Fig. 1shows the effects of dialysis for up to 10 h on the pyridoxal phosphate content of wild type and K87T tryptophan synthase alpha(2)beta(2) complex (A) and beta(2) subunit (B). Pyridoxal phosphate remains largely bound to the wild type alpha(2)beta(2) complex (Fig. 1A, curve1). The pyridoxal phosphate content of the wild type beta(2) subunit decreases from 1.0 to 0.8 mol of pyridoxal phosphate/mol of beta protomer after 10 h (Fig. 1B, curve 1). In contrast, the pyridoxal phosphate content of the K87T alpha(2)beta(2) complex and beta(2) subunit decreases to about 0.4 and 0.1 mol/mol of beta, respectively, after 10 h (Fig. 1, A and B, curves2). Thus, formation of the internal aldimine with lysine 87 in the wild type enzymes stabilizes the enzymes to loss of pyridoxal phosphate upon dialysis. L-Serine reacts with the K87T beta(2) subunit and alpha(2)beta(2) complex to form an external aldimine with pyridoxal phosphate. Formation of this external aldimine markedly stabilizes the K87T alpha(2)beta(2) complex (Fig. 1A, curve3) and K87T beta(2) subunit (Fig. 1B, curve3) to loss of pyridoxal phosphate upon dialysis. The L-serine moiety in the external aldimine may stabilize the pyridoxal phosphate by interacting with residues in the substrate binding site of the beta(2) subunit. This hypothesis is consistent with our previous finding that formation of the external aldimine with L-serine by the K87T alpha(2)beta(2) complex greatly increases the ellipticity band of the coenzyme(20) .


Figure 1: Effect of time of dialysis on the pyridoxal phosphate content of various forms of tryptophan synthase. Solutions of the holo-alpha(2)beta(2) complex (A) and of the holo-beta(2) subunit (B) at 1 mg/ml were dialyzed against Buffer B at 4 °C. Aliquots were analyzed at intervals for protein concentration and for pyridoxal phosphate content. Curve1, wild type; curve2, K87T; curve3, K87T-Ser complex.



Gel Filtration Analysis of Different Forms of Tryptophan Synthase

We have monitored the association state of the alpha and beta(2) subunits from the wild type, the reduced wild type, and the K87T tryptophan synthase by high performance gel filtration on a Superose column with an FPLC system (Fig. 2). The wild type holo-alpha(2)beta(2) complex elutes largely as a single peak at 9.75 ml, whereas the isolated wild type holo-beta(2) subunit and alpha subunit chromatographed separately elute at 10.35 and 12.3 ml, respectively (Fig. 2A). The holo-alpha(2)beta(2) complex has been observed previously as a single species by ultracentrifugation (21, 22) , by sucrose gradient centrifugation (8, 22) and by gel filtration(8, 21, 23) . The wild type apo-alpha(2)beta(2) complex is completely separated into the apo-beta(2) subunit and alpha subunit in this system (data not shown) as demonstrated previously for gel filtration of the apo-alpha(2)beta(2) complex on Sephadex G100 Superfine (15) in the same buffer.


Figure 2: Superose gel filtration of various forms of wild type and mutant (K87T) tryptophan synthase. A, wild type holo-alpha(2)beta(2) complex (1 mg), holo beta(2) subunit (1 mg), and alpha subunit (0.38 mg) were injected in separate runs. B, wild type alpha subunit (0.42 mg) was preincubated for 3 h at 23 °C with approximately equimolar reduced beta(2) subunit (0.66 mg), K87T beta(2) subunit (0.6 mg), or K87T beta(2) subunit plus 50 mML-serine before injection in separate runs. C, wild type alpha subunit was preincubated with 2 eq of beta protomer to give the alphabeta(2) complex; the K87T alpha(2)beta(2) complex was injected as in A.



Mixing the alpha subunit with 2 or more eq of holo-beta protomers results in a stable alphabeta(2) complex that can be observed by sucrose centrifugation (8, 22) or ultracentrifugation(21) . The wild type holo-alphabeta(2) complex elutes at 10.0 ml in our high performance gel filtration system (Fig. 2C). Gel filtration of the K87T holo-alpha(2)beta(2) complex yields two peaks at positions characteristic of the alphabeta(2) complex and of the alpha subunit (Fig. 2C). (^2)The identity of these two species was confirmed by assay of activity in the alpha reaction and by SDS-gel electrophoresis (data not shown). Thus the K87T holo-alpha(2)beta(2) complex partially dissociates under these conditions. Similar results were obtained with a reconstituted K87T holo-alpha(2)beta(2) complex prepared by mixing the K87T holo-beta(2) subunit and 2 eq of wild type alpha subunit (Fig. 2B). As controls, reconstituted alpha(2)beta(2) complexes were also prepared by mixing holo-beta(2) subunit or reduced beta(2) subunit with 2 eq of alpha subunit. The reconstituted reduced alpha(2)beta(2) complex (Fig. 2B) and holo-alpha(2)beta(2) complex (data not shown) each yielded a major peak at the position of alpha(2)beta(2) complex. Gel filtration of the reconstituted K87T holo-alpha(2)beta(2) complex in the presence of L-serine also yielded a major peak at the position of the alpha(2)beta(2) complex. Thus L-serine largely prevents dissociation of the K87T holo-alpha(2)beta(2) complex to alphabeta(2) and alpha subunit under these conditions.

Effects of the K87T Mutation on Association with the alpha Subunit and on Activation of the alpha Subunit

The association reaction between the alpha and beta(2) subunits can be described as shown in .

Fig. 3shows the enzymatic activity of the wild type alpha subunit in the alpha reaction obtained upon titration with increasing amounts of wild type holo-beta(2) subunit, reduced beta(2) subunit, and K87T holo-beta(2) subunit. Titrations with the reduced and K87T holo-beta(2) subunit were also carried out in the presence of L-serine. It is noteworthy that the maximum specific activity of the alpha subunit in the alpha reaction (see S in Table 1) varies significantly when the alpha subunit is complexed with different species of beta(2) subunit. Early studies also observed that alpha(2)beta(2) complexes containing reduced beta(2) subunit from Escherichia coli had 2-fold greater activity than the wild type alpha(2)beta(2) complex in the alpha reaction(23) . It is known that the activity of the alpha subunit is sensitive to the conformational state of the beta(2) subunit because ligands that bind to the active site of the beta(2) subunit alter the kinetics of reaction at the active site of the alpha subunit 25 Å distant(13, 19, 24, 25, 26, 27, 28) . Reduction of pyridoxal phosphate at the active site of the beta(2) subunit (reduced beta(2)) and substitution of threonine for lysine 87 (K87T beta(2)) probably cause alterations in the conformation of the beta(2) subunit that are communicated to the active site of the alpha subunit and alter the activity of the alpha subunit.


Figure 3: Titration of the alpha subunit with various beta(2) subunits. The activity of the alpha subunit (0.88 µM) in the alpha reaction was determined in the presence of various amounts (0-2.64 µM) of the wild type beta(2) subunit (), the reduced beta(2) subunit (box), or the K87T beta(2) subunit (bullet). Data in the presence of 40 mML-serine was determined with various amounts of the reduced beta(2) subunit (circle) or the K87T beta(2) subunit (). Specific activity expressed in units/mg alpha subunit is plotted versus the molar ratio of beta/alpha. Data were analyzed as described in the text and fit to models 1-3 (see Table 2). Derived apparent dissociation constants are shown in Table 2.







The data obtained in Fig. 3and additional data (not shown) for titration with the apo-beta(2) subunit were analyzed using the PC-MLAB program (Civilized Software, Bethesda, MD) and fitted to three possible models (Table 1): 1) the beta(2) subunit binds two alpha subunits with equal affinities (K(d)(1) = K(d)(2); both bound alpha subunits are active); 2) the alpha(2)beta(2) complex is active, whereas the alphabeta(2) complex is inactive; 3) the beta(2) subunit binds two alpha subunits with different affinities (both bound alpha subunits are active). Model 3 gave the best fit (as judged by the sum of squares) for the wild type and reduced beta(2) subunits. (^3)The affinities of the first alpha subunit for the holo-beta(2) subunit are too high to measure by this method (K(d) = <0.01 µM; Table 1). The dashedline in Fig. 3shows the theoretical curve for stoichiometric binding of two alpha subunits to the reduced beta(2) dimer where the K(d) = <0.01 µM for both alpha subunits. Analysis showed that the second alpha subunit binds to the reduced beta(2) subunit and to the wild type holo-beta(2) subunit with K(d) = 0.07-0.08 µM (Table 1). In marked contrast, the K87T holo-beta(2) subunit exhibits a gradual titration curve that indicates much weaker and equal affinities for both alpha subunits with K(d) = 0.4 µM (Table 1). In the presence of L-serine, the K87T holo-beta(2) subunit again exhibits a sharp biphasic titration curve, with the second K(d) = 0.05 µM (Table 1). This result shows that L-serine greatly strengthens the association of the K87T holo-beta(2) subunit with the alpha subunits. Addition of L-serine to the reduced beta(2) subunit has no effect. This is to be expected because the reduced beta(2) subunit does not react with L-serine. Addition of L-serine to the wild type holo-beta(2) subunit in the presence of the alpha subunit and indole-3-glycerol phosphate results in the overall alphabeta reaction which is about 20 times faster than the alpha reaction (data not shown). Association of the alpha and beta(2) subunits under the conditions of the alphabeta reaction is reported to be very tight (K(d) = 0.4 times 10 µM)(8) . The apo-beta(2) subunit exhibits much weaker and equal affinities for both alpha subunits with K(d) = 0.3 µM (Table 1).

Subunit Interchange Experiments

The rate of dissociation of the wild type alpha(2)beta(2) complex has been measured by determining the loss of activity in the beta reaction upon addition of a 20-fold excess inactive reduced beta(2) subunit(9) . (^4)Fig. 4, curve1, shows that the enzymatic activity decreases rapidly to an equilibrium value. Some residual activity is observed at equilibrium since the wild type beta(2) subunit is not completely displaced by the 20-fold excess of reduced beta(2) subunit. The rate of dissociation is greatly reduced in the presence of L-serine (Fig. 4, curve2). Thus, reaction of L-serine with the wild type alpha(2)beta(2) complex tightens association (8) and decreases the rate of dissociation (9) as found previously.


Figure 4: Kinetics of dissociation of the wild type alpha(2)beta(2) complex. Dissociation was initiated by addition of excess reduced beta(2) subunit in the absence (curve1) or presence (curve2) of 40 mML-serine or by addition of the K87T-Ser beta(2) subunit complex in the absence (curve3) or presence (curve4) of 40 mM of L-serine. Activity measured at intervals in the beta reaction is shown relative to the activity of the alpha(2)beta(2) complex alone (see ``Experimental Procedures'').



Our plan to study the rate of dissociation of the wild type alpha(2)beta(2) complex upon addition of excess K87T beta(2) subunit was precluded by our inability to obtain completely serine-free K87T beta(2) subunit. Trial experiments (not shown) indicated that the small fraction (10%) of the K87T beta(2) subunit that bound L-serine (K87T beta(2)-Ser) was much more effective than the serine-free fraction (K87T beta(2)) in binding alpha subunit and causing dissociation of the wild type alpha(2)beta(2) complex. Consequently we determined the rate of dissociation of the wild type alpha(2)beta(2) complex upon addition of the isolated K87T beta(2)-Ser complex in the absence of excess L-serine (curve3) and in the presence of excess L-serine (curve4). Addition of K87T beta(2)-Ser (curve3) results in more rapid dissociation of the wild type alpha(2)beta(2) complex than does addition of the reduced beta(2) subunit (curve1). Thus, K87T beta(2)-Ser binds the alpha subunit more rapidly than does reduced beta(2). Addition of excess L-serine (curve4) tightens the association of the wild type alpha(2)beta(2) complex and decreases the rate of dissociation of the alpha(2)beta(2) complex.

The rate of dissociation of an alpha(2)beta(2) complex containing inactive beta(2) subunit can be measured by determining the increase in activity upon addition of excess wild type beta(2) subunit. This method has been used previously for the reaction of reduced alpha(2)beta(2) complex with excess wild type beta(2) subunit (9) as illustrated in Fig. 5, curve1. We have determined activity in the alphabeta reaction because the free beta(2) subunit has no activity in the alphabeta reaction, whereas the beta(2) subunit does have some activity in the beta reaction that would give a high blank value. Addition of excess wild type beta(2) subunit to the K87T alpha(2)beta(2) complex results in 40% activity within the mixing time (Fig. 5, curve2) followed by a slower increase in activity. When the K87T alpha(2)beta(2)-Ser complex was mixed with wild type beta(2) subunit in the presence of 40 mML-serine (Fig. 5, curve3), the rate of activation was much slower in the first 5 min than in curve2, but full activity was achieved after about 30 min. The reaction of the K87T alpha(2)beta(2)-Ser complex with excess beta(2) subunit (Fig. 5, curve4) showed a decreased rate of activation similar to that in curve3. The results indicate that the first alpha subunit in the K87T alpha(2)beta(2) complex dissociates readily in the absence of L-serine but more slowly in the presence of L-serine. The very slow rate of subunit interchange with the K87T alpha(2)beta(2)-Ser complex (Fig. 5, curve4) implies that the K87T alpha(2)beta(2)-Ser dissociates very slowly. The slow rate of subunit interchange with the K87T alpha(2)beta(2) complex (Fig. 5, curve2) may result from the 3% contamination of the enzyme with L-serine (see ``Experimental Procedures'').


Figure 5: Kinetics of dissociation of the reduced alpha(2)beta(2) complex and K87T alpha(2)beta(2) complex. Excess wild type beta(2) subunit was added to initiate dissociation of the reduced alpha(2)beta(2) complex (curve1), of the K87T alpha(2)beta(2) complex in the absence (curve2) or presence (curve3) of 40 mML-serine, or of the K87T-Ser alpha(2)beta(3) complex (curve4). Activity measured at intervals in the alphabeta reaction is shown relative to the activity of the wild type alpha(2)beta(2) complex (see ``Experimental Procedures'').




DISCUSSION

Our studies of a mutant form of tryptophan synthase, which is unable to form the internal aldimine between beta(2) subunit Lys-87 and pyridoxal phosphate demonstrate that the internal aldimine is very important for stabilizing cofactor binding and subunit association.

Stabilization of Cofactor Binding

Pyridoxal phosphate is removed by dialysis more slowly from the wild type beta(2) subunit and alpha(2)beta(2) complex than from the K87T beta(2) subunit and K87T alpha(2)beta(2) complex which cannot form the internal aldimine (Fig. 1). Thus, formation of the internal aldimine with Lys-87 in the wild type enzymes stabilizes the beta(2) subunit and alpha(2)beta(2) complex to loss of pyridoxal phosphate upon dialysis. Our finding that formation of the external aldimine with L-serine markedly stabilizes the K87T beta(2) subunit and alpha(2)beta(2) complex (Fig. 1) to loss of pyridoxal phosphate upon dialysis suggests that L-serine stabilizes the bound pyridoxal phosphate by interacting with residues in the substrate binding site of the beta(2) subunit and forming a bridge between the N- and C-domain (see below).

Binding of pyridoxal phosphate and pyridoxal phosphate analogues to the wild type beta(2) subunit and alpha(2)beta(2) complex has been studied extensively(29, 30, 31, 32) . Pyridoxal phosphate is bound cooperatively to the apo-beta(2) subunit and noncooperatively to the apo-alpha(2)beta(2) complex. The slow rate of removal of pyridoxal phosphate from the wild type alpha(2)beta(2) complex by dialysis probably results from the very slow reversal of the final isomerization step in pyridoxal phosphate binding(32) . The rate of removal of pyridoxal phosphate from the beta(2) subunit and alpha(2)beta(2) complex by dialysis has not been compared previously. However, we have reported that the pyridoxal phosphate oxime is much more readily removed from the beta(2) subunit than from the alpha(2)beta(2) complex(15) . We have suggested that interaction of the alpha subunit with the beta(2) subunit may stabilize the interaction between the N- and C-domains of the beta(2) subunit and thus slow removal of pyridoxal phosphate and pyridoxal phosphate derivatives that are bound between the two domains(2) .

Stabilization of Subunit Association

-Early studies using sucrose gradient centrifugation showed that association of the alpha and beta(2) subunits of tryptophan synthase was weak in the absence of ligands, intermediate in the presence of pyridoxal phosphate, and strong in the presence of pyridoxal phosphate and L-serine (8) (Table 2). Our gel filtration results (Fig. 2) and activity measurements ( Fig. 3and Table 1) demonstrate weak association between the alpha subunit and a mutant form of the beta(2) subunit (K87T) that binds pyridoxal phosphate as the free aldehyde and not as an internal aldimine. Thus the holo-K87T alpha(2)beta(2) complex resembles the apo-alpha(2)beta(2) complex in having weak subunit association (Table 2). The reaction of L-serine with the K87T alpha(2)beta(2) complex increases the affinity as shown by gel filtration (Fig. 2C) and activity measurements ( Fig. 3and Table 1). The K87T alpha(2)beta(2)-Ser complex thus resembles the holo-alpha(2)beta(2) complex and reduced alpha(2)beta(2) complex in having intermediate subunit association (Table 2).

Subunit interchange experiments show that the rate of dissociation of the holo-alpha(2)beta(2) complex by reduced beta(2) subunit is greatly decreased in the presence of L-serine (Fig. 4, curve 2 versus1). Thus, reaction of L-serine with the wild type alpha(2)beta(2) complex decreases the rate of dissociation (9) and tightens association (strong association in Table 2)(8) . Dissociation of the holo-alpha(2)beta(2) complex by K87T beta(2)-Ser is also greatly reduced in the presence of L-serine (Fig. 4, curve 4 versus3). The similarity of the results with reduced beta(2) and with K87T beta(2)-L-Ser in Fig. 4supports the classification of these two enzymes in the same group (intermediate association) in Table 2. The finding that the K87T alpha(2)beta(2) complex dissociates readily in the absence of L-serine but more slowly in the presence of L-serine (Fig. 5) implies that the external aldimine with L-serine increases association from weak to intermediate (Table 2).

The dissociation constants for various forms of the alpha(2)beta(2) complex ( Fig. 3and Table 1) are apparent dissociation constants since they are measured in the presence of a substrate (indole-3glycerol phosphate) that may alter subunit affinity. Our new methods of analysis of the titration curves shows that most forms of the beta(2) subunit have higher affinity for the first alpha subunit than for the second alpha subunit. This result is consistent with other evidence that the wild type beta(2) subunit binds the alpha subunit with negative cooperativity(9, 33) . The results in Fig. 3and Table 1demonstrate that L-serine increases the affinity of the K87T beta(2) subunit for the alpha subunit by 1-2 orders of magnitude.

Conclusions

Our finding that formation of the external aldimine between pyridoxal phosphate and L-serine increases the affinity of the K87T beta(2) subunit for both pyridoxal phosphate and the alpha subunit suggests that this external aldimine stabilizes pyridoxal phosphate binding by forming a bridge between the N-domain and the C-domain. The proposed interdomain bridge may mimic the internal aldimine bond in the wild type beta(2) subunit and the reduced bond in the reduced beta(2) subunit. Crystallographic studies of the wild type alpha(2)beta(2) complex (7) show that pyridoxal phosphate is bound at the interface between the two domains in each beta protomer and that the external aldimine between L-serine and pyridoxal phosphate is bound between these two domains in the the K87T alpha(2)beta(2)-Ser complex(12) .^1 The interactions of L-serine are largely with the N-domain, whereas those of pyridoxal phosphate are largely with the C-domain.^1

Our previous finding that the K87T alpha(2)beta(2) complex has very weak ellipticity, whereas the external aldimine with L-serine has much greater ellipticity(20) , indicates that pyridoxal phosphate becomes more rigidly oriented when its carbonyl group forms an external aldimine with L-serine. The increased rigidity results from the binding of L-serine to the active site. The reduced alpha(2)beta(2) complex is stabilized by the covalent link between pyridoxal phosphate and Lys-87. The interdomain bridges formed by the internal aldimine with the wild type beta(2) subunit, by reduced phosphopyridoxyl-lysine in the reduced beta(2) subunit, and by the external aldimine with L-serine in the K87T beta(2) subunit may stabilize interaction with the alpha subunit because the the alpha/beta interaction site contains residues from both the N-domain and the C-domain.

The substrate of an enzyme often binds in a cleft between two domains and can be considered to bridge the two domains. Example enzymes include aspartate aminotransferase(34) , citrate synthase(35) , hexokinase(36) , and human alpha-thrombin(37) . Salt bridges and disulfide bonds may also form bridges between two enzyme domains and stabilize the enzymes to unfolding by heat and denaturants.

In conclusion, our work has shed light on aspects of interdomain interaction that stabilize both the beta(2) subunit and its interaction with the alpha subunit. The internal and external aldimine intermediates formed by pyridoxal phosphate stabilize the interaction of pyridoxal phosphate with tryptophan synthase and stabilize the enzyme structure. Recent studies on the thermal inactivation (38) and thermal unfolding (39, 40, 41) (^5)also demonstrate that pyridoxal phosphate stabilizes the beta(2) subunit and alpha(2)beta(2) complex of tryptophan synthase to unfolding and increases the linkage (cooperativity) between unfolding domains in the beta(2) subunit.


FOOTNOTES

*
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§
To whom correspondence and reprint requests should be addressed: NIH, Bldg. 8, Rm. 2A09, Bethesda, MD 20892. Tel.: 301-496-2763; Fax: 301-402-0240.

(^1)
K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies, manuscript in preparation.

(^2)
Addition of pyridoxal phosphate (50 µM) to the buffer slightly reduced the dissociation of the alpha subunit (data not shown).

(^3)
Models 2 and 3 gave equally good fits to the data shown in Fig. 3. However, model 2 (active tetramer) predicts a drop in activity at a higher concentrations of beta(2) subunit due to the disproportionation reaction: alpha(2)beta(2) + beta(2) &cjs0635; 2 alphabeta(2) (inactive). Model 2 is ruled out by our observation that the activity does not drop in assays performed with a high concentration of beta(2) subunit.

(^4)
Measurement of the dissociation rate constant of the holo-alpha(2)beta(2) complex was carried out earlier by use of a mutant alpha subunit, which binds to holo-beta(2) subunit as strongly as wild type alpha subunit, but is inactive in the alpha and alphabeta reactions(8) .

(^5)
D. P. Remeta, E. W. Miles, and A. Ginsburg, manuscript in preparation.


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