Mutation of an Active Site Residue of Tryptophan Synthase (beta -Serine 377) Alters Cofactor Chemistry*

Kwang-Hwan Jhee, Li-hong YangDagger , S. Ashraf Ahmed§, Peter McPhie, Roger Rowlett, and Edith Wilson Milesparallel

From the National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta -Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa ~ 7.7) from a form with a protonated internal aldimine nitrogen (lambda max = 416 nm) to a deprotonated form (lambda max = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta 2 subunit and alpha 2beta 2 complex are pH-independent. The reaction of the S377D alpha 2beta 2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambda max = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP)1-dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1, 2). The enzyme protein directs and restricts the catalytic potential of the bound PLP to provide the substrate and reaction specificity and the enhanced reaction rate of the enzyme (3, 4). Thus, it is important to understand how the protein structure controls the cofactor chemistry and enhances catalytic rates.

Information on how the protein structure controls PLP-dependent reactions is beginning to emerge from x-ray crystallography and from sequence comparisons designed to establish evolutionary relationships (5, 6). One of the most important and best studied interactions between the cofactor and the protein active site of all PLP enzymes is that between the pyridine nitrogen of PLP and an amino acid side chain (see Fig. 1) (7). In L-aspartate aminotransferase (EC 2.6.1.1) (8) and tryptophanase (EC 4.1.99.1),2 this interaction is a hydrogen bond/salt bridge between the N-1 proton of PLP and a negatively charged aspartate side chain. These two enzymes are classed in the alpha  family (5) or fold type I (6). The PLP-binding site of the tryptophan synthase beta  subunit (EC 4.2.1.20),3 a representative of the beta  family or fold type II, has the neutral hydroxyl of Ser377 interacting with PLP N-1 (9, 10). Alanine racemase (EC 5.1.1.1; fold type III) is unique in having a positively charged arginine near N-1 of PLP (7). Although the enzymes in the three different fold types have unrelated three-dimensional structures, the two enzymes in fold type I have related structures. These four enzymes catalyze their different reactions by the pathways illustrated in Fig. 1. The primary reactions of tryptophan synthase and tryptophanase are the synthesis and degradation of L-tryptophan by beta -replacement and beta -elimination reactions, respectively.

In this work, we have used site-directed mutagenesis to change Ser377 to Asp (beta S377D) or Glu (beta S377E) and have determined the effects of these mutations on some kinetic and spectroscopic properties. We have reported briefly (11) that mutation of beta -Ser377 to Ala, Asp, or Glu results in a >100-fold decrease in the rate of conversion of L-serine and indole to tryptophan and that the beta S377D and beta S377E alpha 2beta 2 complexes display some spectral properties similar to those of tryptophanase and aspartate aminotransferase.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals and Buffers-- PLP and beta -chloro-L-alanine hydrochloride were from Sigma. L-Serine was purchased from Fluka. Solutions of beta -chloro-L-alanine hydrochloride were freshly prepared and adjusted to pH 7.8 with sodium hydroxide immediately before use. Buffer A (50 mM sodium Bicine containing 1 mM EDTA at pH 7.8) was used for spectroscopic studies unless otherwise specified. The other buffer used was composed of 50 mM triethanolamine/Bicine (pH 7.8) containing 0.2 M NaCl, 0.2 M CsCl, or 0.2 M KCl.

Mutagenesis-- The expression vector pEBA-10 was used as the template for quick and convenient mutagenesis by megaprimer polymerase chain reaction (PCR) (12). Mutagenic primers used in the construction of the missense mutations were as follows (where base changes are underlined): S377D, 5'-GTC-AAT-CTC-GAT-GGC-CGC-GGA-GT-3'; and S377E; 5'-GTC-AAT-CTC-GAA-GGC-CGC-GGA-GTA-3'. Other primers are described (12). The cloning and expression plasmid for each mutant tryptophan synthase was constructed as described (12). Briefly, each mutagenic primer and PE2 (which contains an XbaI restriction site) were used to amplify the first round of PCR with the pEBA-10 template plasmid using Pfu DNA polymerase (Stratagene). The first PCR fragments were purified and used directly as primers together with the alternate primer PE5 (which contains an SphI restriction site) to amplify the second round of DNA synthesis with the pEBA-10 template plasmid. Deoxyadenosine (dA) was added to the newly amplified second round PCR products by the non-template-dependent activity of Taq polymerase. The second round PCR fragments were purified and directly inserted into the linearized pCRII sequencing plasmid (Invitrogen), which has single 3'-deoxythymidine (dT) residues. After confirmation of the mutated genes by DNA sequencing, the inserted DNA fragment was restricted with SphI and XbaI restriction enzymes (Promega) and ligated into the original parent plasmid (pEBA-10), which had also been digested with SphI and XbaI.

Enzymes-- Escherichia coli CB149 (13), which lacks the trp operon, was used as a host strain for plasmid pEBA-10 (12) that expresses the wild type and mutant beta  subunit forms (S377D and S377E) of the Salmonella typhimurium tryptophan synthase alpha 2beta 2 complex. Cultures of the host harboring wild type or mutant plasmid were grown, and enzyme expression was induced with isopropyl-1-thio-beta -D-galactopyranoside as described (12). Purification of wild type and mutant alpha 2beta 2 complexes utilized crystallization from crude extracts followed by recrystallization (14). The amounts of purified enzymes obtained from 1-liter cultures were 1000 mg (S377D) and 270 mg (S377E). Analysis of the purified enzymes by SDS-polyacrylamide gel electrophoresis reveal that the S377D enzyme contained a lower content of alpha  subunit (~50%) than the wild type alpha 2beta 2 complex and that the S377E enzyme contained only beta  subunit (data not shown). The wild type and S377D beta 2 subunits were obtained by heat precipitation of the alpha  subunit from the alpha 2beta 2 complex (15). Plasmid pEBA-4A8 was used to express the wild type alpha  subunit in E. coli CB149 (12). The alpha  subunit was purified as described (16) with a slight modification; after DEAE-Sephacel column chromatography, all fractions were analyzed by SDS-polyacrylamide gel electrophoresis, and the fractions showing the single band corresponding to the alpha  subunit were combined and concentrated. A 1-liter culture yielded ~1 g of homogeneous alpha subunit. Protein concentrations were determined from the specific absorbance at 278 nm using Acm1% = 6.0 for the holo-alpha 2beta 2 complex, Acm1% = 6.5 for the holo-beta 2 subunit, and Acm1% = 4.4 for the alpha  subunit (15).

Spectroscopic Methods-- Absorption spectra were measured in a Hewlett-Packard 8452 diode array spectrophotometer thermostatted at 25 °C. Circular dichroism measurements (mean residue ellipticity in degrees cm2/dmol) were made in a Jasco J-500C spectrophotometer equipped with a DP-500N data processor (Japan Spectroscopic Co., Easton, MD).

Data Analysis-- The effects of pH on absorbance at 416 nm were analyzed by Equation 1,
E<UP>H<SUP>+</SUP></UP> ⇌ E+<UP>H<SUP>+</SUP></UP> (Eq. 1)
where EH+ and E represent the enzyme with a protonated (416 nm species) and deprotonated (334-338 nm species) internal Schiff base, respectively. The pKa value for the dissociation of EH+ was derived from nonlinear least squares fit to Equation 2,
A=A<SUB><UP>min</UP></SUB>+<FR><NU>(A<SUB><UP>max</UP></SUB>−A<SUB><UP>min</UP></SUB>)</NU><DE>(1+K<SUB>a</SUB>/[<UP>H<SUP>+</SUP></UP>])</DE></FR> (Eq. 2)
where A is the absorbance at 416 nm and Amin and Amax are the minimum and maximum absorbance values, respectively.

The interaction of the alpha  and beta  subunits was characterized by measuring the absorbance maxima at 504 nm in the presence of L-tryptophan as a function of alpha  subunit concentration. The data were modeled assuming that the alpha  and beta  subunits associate in a noncooperative fashion. Under this assumption, the alpha -beta interaction can be simply modeled according to Equation 3.
K<SUB>d</SUB>=[&agr;][&bgr;]/[&agr;&bgr;] (Eq. 3)
The position of the equilibrium of Equation 3 can be monitored by absorbance measurements, where the maximum absorbance A A0 + (Amax - A0)falpha beta , falpha beta  = [alpha beta ]/[beta ]tot, A0 is the maximum absorbance of the beta 2 subunit alone with L-tryptophan (S377D and S377E), and Amax is the intrinsic maximum absorbance of the alpha 2beta 2 complex with L-tryptophan. The concentration of [alpha beta ] at any given [alpha ]tot/[beta ]tot ratio can be solved explicitly from Equation 4,
K<SUB>d</SUB>=<FR><NU>(f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB>−[&agr;&bgr;])([&bgr;]<SUB><UP>tot</UP></SUB>−[&agr;&bgr;])</NU><DE>[&agr;&bgr;]</DE></FR> (Eq. 4)
where falpha  = [alpha ]tot/[beta ]. The unique solution for [alpha beta ] is described by Equation 5,
[&agr;&bgr;]=<FR><NU>([&bgr;]<SUB><UP>tot</UP></SUB>+f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB>+K<SUB>d</SUB>)−<RAD><RCD>([&bgr;]<SUB><UP>tot</UP></SUB>+f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB>+K<SUB>d</SUB>)<SUP>2</SUP>−4f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB><SUP><UP>2</UP></SUP></RCD></RAD></NU><DE>2</DE></FR> (Eq. 5)
and the variation of maximum absorbance (A) with the fraction of added alpha  subunit (falpha ) is given by Equation 6.
A=A<SUB>0</SUB>+(A<SUB><UP>max</UP></SUB>−A<SUB>0</SUB>)
·<FR><NU>([&bgr;]<SUB><UP>tot</UP></SUB>+f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB>+K<SUB>d</SUB>)−<RAD><RCD>([&bgr;]<SUB><UP>tot</UP></SUB>+f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB>+K<SUB>d</SUB>)<SUP>2</SUP>−4f<SUB>&agr;</SUB>[&bgr;]<SUB><UP>tot</UP></SUB><SUP>2</SUP></RCD></RAD></NU><DE>2[&bgr;]<SUB><UP>tot</UP></SUB></DE></FR>
The dissociation constants for the alpha  and beta  subunits in the presence of L-tryptophan were obtained from Equation 6 and are termed apparent dissociation constants (Kd(alpha beta )) because they were determined in the presence of ligands including L-tryptophan and cations.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

We have altered the active site of the tryptophan synthase beta  subunit by changing a residue near the pyridine nitrogen (N-1) of PLP from a neutral Ser to a negatively charged Asp or Glu as found in aspartate aminotransferase and tryptophanase (Fig. 1). The engineered mutant beta  subunits were expressed in high yield by a vector that also expresses the wild type alpha  subunit. Purification of the mutant beta  subunits by a method that has been used to purify the wild type alpha 2beta 2 complex (12) and beta 2 subunit (14, 16) resulted in a partial loss of alpha  subunit from the S377D beta  subunit and a complete loss of alpha  subunit from the S377E beta  subunit, as described under "Experimental Procedures." The results suggest that association of the S377D and S377E beta  subunits with the alpha subunit is weaker than that of the wild type beta  subunit, as demonstrated below.


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Fig. 1.   PLP-binding sites and intermediates in reactions catalyzed by four PLP enzymes. PLP forms an internal aldimine (E) with the epsilon -amino group of the enzyme lysine; N-1 of PLP interacts with residue X, which is Asp in aspartate aminotransferase (AspAT) and tryptophanase (TPase), Ser in tryptophan synthase (TSase), and Arg in Ala racemase (see the Introduction). The reaction catalyzed by each enzyme proceeds through a series of PLP intermediates, where E-S is the enzyme-substrate intermediate and E-Q1 and E-Q2 are quinonoid intermediates (see Fig. 3 for structures). Tryptophan synthase and tryptophanase catalyze the beta -elimination of OH- from L-serine to form E-AA, the aminoacrylate intermediate, which either is hydrolyzed to pyruvate and NH3 or reacts with indole (IND) to form E-Q2, followed by protonation to form E-Trp, the enzyme-product complex. The different pathways illustrate the reaction specificity of each enzyme. It is clear that the efficacy of each enzyme lies not only in its ability to accelerate the required reaction at each stage, but as far as possible to prevent all the other alternatives (4). PMP, pyridoxamine phosphate.

Effects of pH on the Absorbance and Ellipticity Properties of the S377D beta 2 Subunit and alpha 2beta 2 Complex-- The absorption spectra of the pyridoxal phosphate cofactor of the tryptophan synthase beta 2 subunit and alpha 2beta 2 complex from E. coli (17) and of the alpha 2beta 2 complex from S. typhimurium (18) are pH-independent between pH 6 and 10. The absorption spectra of the S377A beta 2 subunit and alpha 2beta 2 complex are also pH-independent (11). In contrast, the absorption spectra of the S377D beta 2 subunit (Fig. 2A) and of the S377E beta 2 subunit (data not shown) exhibit two pH-dependent absorption bands with maxima at 334 and 416 nm. The sharp isosbestic point indicates that there are only two significantly populated species involved.


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Fig. 2.   Effect of pH on the absorbance and ellipticity properties of the S377D beta 2 subunit and alpha 2beta 2 complex. A, absorption spectra of the S377D beta 2 subunit (1 mg/ml in Buffer A) made at 25 °C at the indicated pH values. B, pH dependence of the absorbance of the S377D beta 2 subunit at 334 nm (open circle ) and 416 nm (square ) and of the S377D alpha 2beta 2 complex at 334 nm (bullet ) and 416 nm (black-square) measured in Buffer A in the presence of a 3-fold excess of alpha  subunit. Derived pKa values are 7.63 ± 0.06 for the S377D beta 2 subunit and 7.89 ± 0.08 for the S377D alpha 2beta 2 complex. C, CD spectra of the S377D alpha 2beta 2 complex made at the indicated pH values under the conditions described for B.

Analysis of plots of absorbance against pH at these wavelengths (Fig. 2B) shows pKa values of 7.63 ± 0.06 for this ionization for the S377D beta 2 subunit and of 7.89 ± 0.08 for the S377D alpha 2beta 2 complex. The finding that the pKa value for the S377E beta 2 subunit (7.78; data not shown) is 0.15 pH units higher than that for the S337D beta 2 subunit (7.63; Fig. 2B) is consistent with the higher pKa value for Glu (4.5) compared with Asp (4.1) in polypeptides and uncharged derivatives of Asp and Glu (19). The CD spectra of the S377D alpha 2beta 2 complex show that the ellipticity band with a maximum at 416 nm is also pH-dependent (Fig. 2C). The absorption band centered at 334 nm appears to be optically inactive.

Spectroscopic Properties of the S377D alpha 2beta 2 Complex: Accumulation of Quinonoid Intermediates-- The wild type alpha 2beta 2 complex and beta 2 subunit catalyze beta -replacement reactions with L-serine and indole or beta -mercaptoethanol that proceed through a series of PLP intermediates (Fig. 1), which have characteristic absorption spectra (20). Two quinonoid-type intermediates occur in this pathway, the first (E-Q1) after removal of the alpha -proton of L-serine and the second (E-Q2) after the beta -addition of a nucleophile to the aminoacrylate intermediate (E-AA). E-Q1 has been observed in rapid kinetic studies of the alpha 2beta 2 complex as a transitory intermediate with maximum absorbance at 460 nm (21), but does not accumulate under equilibrium conditions. E-Q2 accumulates as a stable intermediate under steady-state conditions in reactions of the wild type alpha 2beta 2 complex with L-serine and nucleophiles, including beta -mercaptoethanol and indole (17), and with the product, L-tryptophan (22, 23).

Mutation of beta -Ser377 to Ala, Asp, or Glu results in a >100-fold decrease in the rate of conversion of L-serine and indole to tryptophan by the mutant alpha 2beta 2 complexes (11).4 Nevertheless, addition of substrates to the S377D alpha 2beta 2 complex in the presence of different monovalent cations results in formation of new absorption bands near 500 nm that exhibit maximum absorbance within 2.5-9 min (Fig. 3 and Table I). The pronounced band that accumulates in the reaction with L-serine in the presence of Cs+ can be attributed to E-Q1. More intense bands are observed in the reactions with L-tryptophan or with L-serine and beta -mercaptoethanol. These bands can be attributed to E-Q2 and exhibit absorption maxima at longer wavelengths (Table I) than quinonoid bands observed with the wild type alpha 2beta 2 complex. The structure of the quinonoid (E-Q) is shown in Fig. 3. The tryptophan quinonoid has absorbance maxima at 476 and 504 nm with the wild type and S377D alpha 2beta 2 complexes, respectively (Table I), whereas the quinonoid formed from L-serine and beta -mercaptoethanol exhibits absorbance maxima at 468 and 508 nm with the wild type and mutant enzymes, respectively. The L-tryptophan quinonoid has a much greater maximum absorbance with the S377D alpha 2beta 2 complex (epsilon 504 nm = 49.2 mM-1 cm-1) than with the wild type alpha 2beta 2 complex (epsilon 476 nm = 1.4 mM-1 cm-1) (23) (Table I).


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Fig. 3.   Structure and absorption spectra of quinonoid intermediates formed by the S377D alpha 2beta 2 complex in the presence of substrates and monovalent cations. Spectra of the S377D beta  subunit (23.3 µM) in the presence of a 3-fold molar excess of alpha  subunit were recorded at 25 °C in 50 mM triethanolamine/Bicine (pH 7.8) ~6 min after addition of 10 mM L-tryptophan and 0.17 M CsCl (trace A); 50 mM L-serine, 50 mM 2-mercaptoethanol, and 0.17 M CsCl (trace B); 50 mM L-serine and 0.17 M CsCl (trace C); 10 mM beta -chloro-L-alanine and 0.17 M CsCl (trace D); or 50 mM L-serine and 0.17 M NH4Cl (trace E). Values of lambda max and molar absorptivity coefficients are collected in Table I.

                              
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Table I
Spectroscopic properties of quinonoid intermediates formed by the S377D alpha 2beta 2 complex
Absorption spectra were recorded as described in the legend to Fig. 3 at intervals after mixing the S377D beta  subunit (23.3 µM) in the presence of a 3-fold excess of alpha  subunit with substrate: L-tryptophan (10 mM), beta -chloro-L-alanine (10 mM), or L-serine (50 mM) in the presence or absence of beta -mercaptoethanol (50 mM) and in the presence of 0.17 M NaCl, CsCl, KCl, or NH4Cl. The maximum absorptivity (epsilon max) of each quinonoid band at the maximum wavelength (lambda max) is given at the time at which maximum absorbance was obtained (tmax). The half-time of disappearance of each quinonoid band (t1/2) is also shown.

The quinonoid bands formed differ in stability (Table I). The half-times for disappearance range from 3 min for the L-serine intermediate in the presence of K+ to 24 h for the L-tryptophan quinonoid in the presence of Cs+ (Table I). The disappearance of the L-serine intermediate probably reflects either the slow conversion to pyruvate by the very low catalytic activity of the S377D alpha 2beta 2 complex or the occurrence of irreversible inactivation of the S377D alpha 2beta 2 complex by a reaction intermediate or both (11).

alpha Subunit Is Required for Tryptophan Quinonoid Formation by the S377D beta 2 Subunit-- The S377D beta 2 subunit alone forms no quinonoid intermediate from L-tryptophan in the presence of Cs+ (Fig. 4). Addition of 0.5-3.5 molar eq of alpha  subunit results in formation of increasing amounts of the quinonoid intermediate. The inset shows a plot of the molar absorbance against the alpha /beta subunit molar ratio. Fit of the data to Equations 3-6 under "Experimental Procedures" gives values of the apparent dissociation constant Kd(alpha beta ) = 7.0 ± 0.2 µM and of 43,000 ± 160 M-1 cm-1 for the maximum absorptivity at 504 nm. (The higher value of the maximum absorptivity at 504 nm, 49,200 M-1 cm-1, reported in Table I was obtained with more freshly prepared enzyme.) Titration of the S377E beta 2 subunit with the alpha  subunit by the same method gave values of Kd(alpha beta ) = 32 ± 3.6 µM and of 1750 ± 60 M-1 cm-1 for the maximum absorptivity. Thus, association of the S377E beta  subunit with the alpha  subunit is weaker than that of the S377D beta  subunit, consistent with the greater loss of alpha  subunit during purification of the S377E beta  subunit from extracts containing alpha subunit.


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Fig. 4.   Effect of alpha  subunit concentration on formation of the tryptophan quinonoid by the S377D beta 2 subunit. Absorption spectra of the S377D beta 2 subunit (23.3 µM in 50 mM triethanolamine/Bicine (pH 7.8) containing 0.2 M CsCl and 10 mM L-tryptophan) were determined at 25 °C in the presence of the indicated alpha  subunit molar ratio. The inset shows a plot of the millimolar absorptivity at 504 nm versus the alpha /beta subunit molar ratio. Values of Kd(alpha beta ) = 7.04 ± 0.16 µM and of 43,116 ± 156 M -1 cm-1 for the maximum molar absorptivity were calculated from these data (see "Experimental Procedures"). Titration of the S377E beta 2 subunit with the alpha  subunit by the same method gave values of Kd(alpha beta ) = 32.2 ± 3.55 µM and of 1747 ± 60 M-1 cm-1 for the maximum molar absorptivity coefficient.

We cannot directly compare the dissociation constant for the S377D beta  subunit with that for the wild type beta  subunit because the constants have not been determined under the same conditions. The apparent dissociation constant for the S377D beta  subunit in the presence of L-tryptophan and Cs+ (Kd(alpha beta ) = 7.0 ± 0.16 µM) is 137-fold higher than the value of Kd(alpha beta ) = 0.051 ± 0.005 µM determined for the wild type alpha  and beta  subunits from measurements of enzymatic activity in the presence of Cs+ in the reaction of L-serine with indole to form L-tryptophan (46) and 3.5-fold higher than the value of Kd(alpha beta ) = 2 µM determined from sedimentation equilibrium in the absence of ligands (24). The presence of L-serine is known to tighten the association between the alpha  and beta  subunits (25). The weaker association of the S377D beta  subunit with the alpha  subunit is consistent with loss of approximately one alpha  subunit during purification.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The experiments described above were aimed at assessing the functional role of beta -Ser377 in the PLP-binding site of tryptophan synthase and the effects of replacing this residue with a negatively charged Asp or Glu residue, which is found in several other PLP-dependent enzymes including aspartate aminotransferase and tryptophanase (see Fig. 1). Our most important results are that the mutant enzymes display pH-dependent spectral changes and exhibit enhanced formation of quinonoid intermediates. We discuss these results in relation to the chemistry of PLP and PLP-dependent enzymes.

All known PLP-dependent enzymes bind PLP as an internal aldimine (Schiff base) with the epsilon -amino group of an enzyme lysyl residue (see E in Fig. 1). Although the equilibria and absorption spectra of PLP Schiff bases have been investigated in model systems (26), binding of PLP to the active site of an enzyme affects the electronic and spectral properties of the PLP derivatives. The interaction of a residue X with the pyridine nitrogen (N-1) of PLP influences the electronic state of the cofactor, the equilibrium distribution between the different reaction intermediates in Fig. 1, and the pathway of catalysis. Effects of a negatively charged carboxylate (X = Asp or Glu) near N-1 of PLP are discussed here with reference to the probable structures of the low and high pH forms of internal aldimines shown in Fig. 5.


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Fig. 5.   Probable structures of the internal aldimines of the low (structure I) and high (structure IIa or IIb) pH forms of the tryptophan synthase S377D mutant. See "Discussion" for details.

A Nearby Carboxylate Raises the pKa of N-1 of PLP-- Interaction of the carboxylate stabilizes the proton on N-1 (Ha) (see Fig. 5) and increases the pKa by 2-5 units (7, 27). Although the position of the proton Ha cannot be determined by crystallography, semiempirical calculations of absorption spectra of aspartate aminotransferase suggest that the N-1-Asp222 pair may be present in both the charged and neutral states, i.e. Ha may be located on the carboxyl or on N-1 (27).

Although the electron-accepting properties of the pyridine ring are enhanced by protonation (7), the presence of a negatively charged Asp near N-1 would reduce the ability of the pyridine ring to attract electrons. Semiempirical calculations of the electronic absorption spectra of PLP derivatives of mitochondrial aspartate aminotransferase (in which X = Asp222) have been carried out using information from x-ray data (28). Calculations of the molecular orbital energy level for PLP in the presence of various protein residues showed that inclusion of Asp222 alone raises the molecular orbital energies by ~3 eV relative to those of PLP. However, inclusion of groups hydrogen-bonded to Asp222 (a water molecule and the His143-Ser139 hydrogen-bonded pair) substantially lowers the molecular orbital energies. Thus, in the case of aspartate aminotransferase, the charge density on active site Asp is modulated by hydrogen-bonding to other groups. The crystal structure of the wild type tryptophan synthase alpha 2beta 2 complex shows the presence of no water molecules or other residues that might interact with the carboxylate that has been introduced in the beta S377D mutant enzyme. It is possible that the low activities observed for the mutant enzymes (11)4 result from the absence of residues that modulate the charge density of the introduced carboxylate.

A Carboxylate near N-1 of PLP Reduces the pKa of the Schiff Base Nitrogen-- Although the pKa of the Schiff base nitrogen is usually well over 11 for model Schiff bases with PLP, a value close to 9.6 is observed for the Schiff base between valine and N-methyl-PLP, which has a positive charge on N-1 (29). This result provides evidence that the protonated state of the pyridine nitrogen (N-1) reduces the pKa of the Schiff base nitrogen by ~2.5 units. The pH-dependent changes observed with tryptophanase and aspartate aminotransferase have been attributed to dissociation of the proton (Hb) on the Schiff base nitrogen (see Fig. 5). Tryptophanase undergoes a pH-dependent change (pKa = 7.2) from a protonated form (lambda max = 420 nm) to a deprotonated form (lambda max = 337 nm) in the presence of K+ (30). Aspartate aminotransferase exhibits a pH-dependent conversion (pKa = 6.7) from a protonated internal form (lambda max = 430 nm) to a deprotonated form (lambda max = 358 nm) (31, 32). The finding that mutation of aspartate aminotransferase Asp222 to Ala (D222A) or Asn (D222N) yields enzymes with pH-independent absorption spectra provides evidence that Asp222 is indeed responsible for reducing the pKa of the Schiff base nitrogen (31, 33).

Although the spectra of tryptophan synthase are pH-independent (18, 34), the absorption spectra of the mutant enzymes engineered in this work are pH-dependent (Fig. 2). These results provide evidence that the engineered beta -Asp377 or beta -Glu377 interacts with the protonated N-1 of PLP and lowers the pKa of the imine nitrogen of the internal aldimine, as proposed for aspartate aminotransferase (31, 32). Our finding that the wild type and S377A beta 2 subunits (11) have pH-independent spectra similar to those of the D222A mutant of aspartate aminotransferase gives further evidence that the charge on the residue near N-1 of PLP affects the electron distribution of the cofactor.

Probable structures for the high and low pH forms of the S377D and S377E mutant enzymes are shown in Fig. 5. The low pH form (structure I) with lambda max = 416 nm has two protons, Ha on N-1 and Hb on the Schiff base nitrogen. Ha may reside on the carboxylate some of the time as discussed above. The high pH form (structures IIa and IIb) with lambda max = 334 nm results from loss of one proton. The structure could be either a neutral form (structure IIa), resulting from dissociation of Ha and transfer of Hb to the phenolic hydroxyl, or a dipolar ionic form (structure IIb), resulting from dissociation of Hb. The observed lambda max = 334 nm is consistent with that of the neutral enolimine form of the Schiff base, which is in equilibrium with the resonance-stabilized ketoenamine form in the wild type tryptophan synthase (35). The enolimine form predominates following a thermally induced reversible conformational transition of the beta 2 subunit (35). The high pH form of tryptophanase also has lambda max = 337 nm, consistent with an enolimine structure (36-38). The dipolar ionic form (structure IIb) is the structure that has been suggested for the high pH form of aspartate aminotransferase, which has lambda max = 360 nm (39). Consequently, the enolimine form (structure IIa) is the more likely structure for tryptophan synthase. The locations of the protons shown in Fig. 5 cannot be established by crystallography, but can, in favorable cases, be determined by 1H NMR spectroscopy (40). Hb dissociates around a pKa value of 6.4 with aspartate aminotransferase (40).

A Carboxylate near N-1 of PLP Promotes Quinonoid Formation-- According to the generally accepted mechanism of catalysis by PLP enzymes, formation of the initial enzyme-substrate intermediate (E-S in Fig. 1) is followed by withdrawal of an electron pair from the alpha -carbon into the pyridine ring (1). The electron-accepting properties of the pyridine ring are enhanced by protonation of the ring nitrogen (N-1), as discussed above. If the ring nitrogen is protonated, cleavage of the C-alpha -H bond leads directly to the quinonoid E-Q1 in Fig. 1 (for structure, see Fig. 3). Evidence that the presence of a proton or methyl group on N-1 of PLP facilitates quinonoid formation is provided by studies of quinonoid formation in model and enzymatic systems with pyridoxal and N-methylpyridoxal (41). Investigations of the spectra of quinonoids derived from O-methyl-PLP and PLP suggest that the proton may have migrated from the Schiff base nitrogen to O-3' (41, 42), as shown in the structure in Fig. 3.

Conclusions-- We conclude that replacing tryptophan synthase beta  subunit Ser377 with a negatively charged Asp or Glu in the PLP-binding site alters the electron distribution in the PLP derivatives. Some of the spectroscopic properties of the mutant enzymes become similar to those of tryptophanase and aspartate aminotransferase, enzymes that also have an Asp residue near N-1 of PLP, but have very different overall structures.

    ACKNOWLEDGEMENTS

We thank Drs. Esmond E. Snell, David E. Metzler, and Dagmar Ringe for reading different drafts of this manuscript and for helpful comments and discussions.

    FOOTNOTES

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

Dagger Present address: Dept. of Biochemistry, Uniformed Services University of Health Sciences, Bldg. B, Rm. 4047, 4301 Jones Bridge Rd., Bethesda, MD 20814.

§ Present address: Dept. of Immunology and Molecular Biology, Toxicology Division, USAMRID, Fort Detrick, Frederick, MD 20702.

Present address: Dept. of Chemistry, Colgate University, 13 Oak Dr., Hamilton, NY 13346-1398.

parallel To whom correspondence and reprint requests should be addressed: Lab. of Biochemistry and Genetics, NIDDK, Bldg. 8, Rm. 225, 8 Center Dr. MSC 0830, Bethesda, MD 20892-0830. Tel.: 301-496-2763; Fax: 301-402-0240; E-mail: EdithM{at}intra.niddk.nih.gov.

1 The abbreviations used are: PLP, pyridoxal phosphate; Bicine, N,N-bis(2-hydroxyethyl)glycine; PCR, polymerase chain reaction.

2 Preliminary crystallographic data have been reported for tryptophanase (43). The structure of tryptophanase is very similar to that of tyrosine phenol-lyase, which has been reported in more detail (44, 45). Asp223 interacts with N-1 of PLP in the structure of tryptophanase from Proteus vulgaris.

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

4 The activity of the S377D alpha 2beta 2 complex is also very low in the reaction with beta -chloro-L-alanine and indole and in beta -elimination reactions with L-serine or beta -chloro-L-alanine. The activities of the S377D alpha 2beta 2 complexes are rather insensitive to pH between 7 and 9.5 (K.-H. Jhee, unpublished results).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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