Portal de Periódicos da CAPES

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

1998; Elsevier BV; Volume: 273; Issue: 19 Linguagem: Inglês

10.1074/jbc.273.19.11417

1083-351X

Kwang-Hwan Jhee, Lihong Yang, Salman Ahmed, Peter McPhie, Roger S. Rowlett, Edith Wilson Miles,

Amino Acid Enzymes and Metabolism

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 (β-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 (pK a ∼ 7.7) from a form with a protonated internal aldimine nitrogen (λmax = 416 nm) to a deprotonated form (λmax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase β2 subunit and α2β2 complex are pH-independent. The reaction of the S377D α2β2 complex withl-serine, l-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (λmax = 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 pK a of the internal aldimine nitrogen and promoting formation of quinonoid intermediates. 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 (β-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 (pK a ∼ 7.7) from a form with a protonated internal aldimine nitrogen (λmax = 416 nm) to a deprotonated form (λmax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase β2 subunit and α2β2 complex are pH-independent. The reaction of the S377D α2β2 complex withl-serine, l-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (λmax = 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 pK a of the internal aldimine nitrogen and promoting formation of quinonoid intermediates. An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP) 1The abbreviations used are: PLP, pyridoxal phosphate; Bicine,N,N-bis(2-hydroxyethyl)glycine; PCR, polymerase chain reaction. -dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1Metzler D.E. Ikawa M. Snell E.E. J. Am. Chem. Soc. 1954; 76: 648-652Crossref Scopus (471) Google Scholar, 2Jencks W.P. Catalysis in Chemistry and Enzymology. McGraw-Hill Book Co., New York1969: 133-146Google Scholar). 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 (3Snell E.E. Christen P. Metzler D.E. Transaminases. John Wiley & Sons, Inc., New York1985: 19-35Google Scholar, 4John R.A. Biochim. Biophys. Acta. 1995; 1248: 81-96Crossref PubMed Scopus (333) Google Scholar). 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 (5Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (347) Google Scholar, 6Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (343) Google Scholar). 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) (7Shaw J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (257) Google Scholar). In l-aspartate aminotransferase (EC 2.6.1.1) (8Kirsch J.F. Eichele G. Ford G.C. Vincent M.G. Jansonius J.N. Gehring H. Christen P. J. Mol. Biol. 1984; 174: 497-525Crossref PubMed Scopus (428) Google Scholar) and tryptophanase (EC4.1.99.1), 2Preliminary crystallographic data have been reported for tryptophanase (43Dementieva I.S. Zakomirdina L.N. Sinitzina N.I. Antson A.A. Wilson K.S. Isupov M.N. Lebedev A.A. Harutyunyan E. J. Mol. Biol. 1994; 235: 783-786Crossref PubMed Scopus (9) Google Scholar). The structure of tryptophanase is very similar to that of tyrosine phenol-lyase, which has been reported in more detail (44Antson A.A. Demidkina T.V. Gollnick P. Dauter Z. Von Tersch R.L. Long J. Berezhnoy S.N. Phillips R.S. Harutyunyan E.H. Wilson K.S. Biochemistry. 1993; 32: 4195-4206Crossref PubMed Scopus (131) Google Scholar, 45Sundararaju B. Antson A.A. Phillips R.S. Demidkina T.V. Barbolina M.V. Gollnick P. Dodson G.G. Wilson K.S. Biochemistry. 1997; 36: 6502-6510Crossref PubMed Scopus (74) Google Scholar). Asp223 interacts with N-1 of PLP in the structure of tryptophanase from Proteus vulgaris. 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 α family (5Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (347) Google Scholar) or fold type I (6Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (343) Google Scholar). The PLP-binding site of the tryptophan synthase β subunit (EC4.2.1.20), 3The term β2 subunit is used for the isolated enzyme in solution; β subunit is used for the enzyme in the α2β2 complex or to describe a specific residue in the β subunit. a representative of the β family or fold type II, has the neutral hydroxyl of Ser377 interacting with PLP N-1 (9Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar, 10Rhee S. Parris K.D. Hyde C.C. Ahmed S.A. Miles E.W. Davies D.R. Biochemistry. 1997; 36: 7664-7680Crossref PubMed Scopus (144) Google Scholar). Alanine racemase (EC 5.1.1.1; fold type III) is unique in having a positively charged arginine near N-1 of PLP (7Shaw J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (257) Google Scholar). 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 β-replacement and β-elimination reactions, respectively. In this work, we have used site-directed mutagenesis to change Ser377 to Asp (βS377D) or Glu (βS377E) and have determined the effects of these mutations on some kinetic and spectroscopic properties. We have reported briefly (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar) that mutation of β-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 βS377D and βS377E α2β2 complexes display some spectral properties similar to those of tryptophanase and aspartate aminotransferase. PLP and β-chloro-l-alanine hydrochloride were from Sigma.l-Serine was purchased from Fluka. Solutions of β-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 mmEDTA at pH 7.8) was used for spectroscopic studies unless otherwise specified. The other buffer used was composed of 50 mmtriethanolamine/Bicine (pH 7.8) containing 0.2 m NaCl, 0.2m CsCl, or 0.2 m KCl. The expression vector pEBA-10 was used as the template for quick and convenient mutagenesis by megaprimer polymerase chain reaction (PCR) (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar). 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 (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar). The cloning and expression plasmid for each mutant tryptophan synthase was constructed as described (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar). 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 anSphI 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 Taqpolymerase. 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. Escherichia coli CB149 (13Kawasaki H. Bauerle R. Zon G. Ahmed S.A. Miles E.W. J. Biol. Chem. 1987; 262: 10678-10683Abstract Full Text PDF PubMed Google Scholar), which lacks the trp operon, was used as a host strain for plasmid pEBA-10 (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar) that expresses the wild type and mutant β subunit forms (S377D and S377E) of the Salmonella typhimurium tryptophan synthase α2β2 complex. Cultures of the host harboring wild type or mutant plasmid were grown, and enzyme expression was induced with isopropyl-1-thio-β-d-galactopyranoside as described (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar). Purification of wild type and mutant α2β2 complexes utilized crystallization from crude extracts followed by recrystallization (14Miles E.W. Kawasaki H. Ahmed S.A. Morita H. Morita H. Nagata S. J. Biol. Chem. 1989; 264: 6280-6287Abstract Full Text PDF PubMed Google Scholar). 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 α subunit (∼50%) than the wild type α2β2 complex and that the S377E enzyme contained only β subunit (data not shown). The wild type and S377D β2 subunits were obtained by heat precipitation of the α subunit from the α2β2 complex (15Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Crossref PubMed Scopus (95) Google Scholar). Plasmid pEBA-4A8 was used to express the wild type α subunit inE. coli CB149 (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar). The α subunit was purified as described (16Yang X.-J. Ruvinov S.B. Miles E.M. Protein Expression Purif. 1992; 3: 347-354Crossref PubMed Scopus (17) Google Scholar) 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 α subunit were combined and concentrated. A 1-liter culture yielded ∼1 g of homogeneous α subunit. Protein concentrations were determined from the specific absorbance at 278 nm using A cm1% = 6.0 for the holo-α2β2 complex,A cm1% = 6.5 for the holo-β2 subunit, andA cm1% = 4.4 for the α subunit (15Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Crossref PubMed Scopus (95) Google Scholar). 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). The effects of pH on absorbance at 416 nm were analyzed by Equation 1, EH+⇌E+H+Equation 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 pK a value for the dissociation of EH+ was derived from nonlinear least squares fit to Equation 2, A=Amin+(Amax−Amin)(1+Ka/[H+])Equation 2 where A is the absorbance at 416 nm andA min and A max are the minimum and maximum absorbance values, respectively. The interaction of the α and β subunits was characterized by measuring the absorbance maxima at 504 nm in the presence ofl-tryptophan as a function of α subunit concentration. The data were modeled assuming that the α and β subunits associate in a noncooperative fashion. Under this assumption, the α-β interaction can be simply modeled according to Equation 3. Kd=[α][β]/[αβ]Equation 3 The position of the equilibrium of Equation 3 can be monitored by absorbance measurements, where the maximum absorbanceA = A 0 + (A max −A 0)f αβ,f αβ = [αβ]/[β]tot,A 0 is the maximum absorbance of the β2 subunit alone with l-tryptophan (S377D and S377E), and A max is the intrinsic maximum absorbance of the α2β2 complex withl-tryptophan. The concentration of [αβ] at any given [α]tot/[β]tot ratio can be solved explicitly from Equation 4, Kd=(fα[β]tot−[αβ])([β]tot−[αβ])[αβ]Equation 4 where f α = [α]tot/[β]. The unique solution for [αβ] is described by Equation 5, [αβ]=([β]tot+fα[β]tot+Kd)−([β]tot+fα[β]tot+Kd)2−4fα[β]tot22Equation 5 and the variation of maximum absorbance (A) with the fraction of added α subunit (f α) is given by Equation 6. A=A0+(Amax−A0) ·([β]tot+fα[β]tot+Kd)−([β]tot+fα[β]tot+Kd)2−4fα[β]tot22[β]totEquation 6 The dissociation constants for the α and β subunits in the presence of l-tryptophan were obtained from Equation 6 and are termed apparent dissociation constants (K d (αβ)) because they were determined in the presence of ligands includingl-tryptophan and cations. We have altered the active site of the tryptophan synthase β 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 β subunits were expressed in high yield by a vector that also expresses the wild type α subunit. Purification of the mutant β subunits by a method that has been used to purify the wild type α2β2 complex (12Yang L.-h. Ahmed S.A. Miles E.W. Protein Expression Purif. 1996; 8: 126-136Crossref PubMed Scopus (41) Google Scholar) and β2 subunit (14Miles E.W. Kawasaki H. Ahmed S.A. Morita H. Morita H. Nagata S. J. Biol. Chem. 1989; 264: 6280-6287Abstract Full Text PDF PubMed Google Scholar, 16Yang X.-J. Ruvinov S.B. Miles E.M. Protein Expression Purif. 1992; 3: 347-354Crossref PubMed Scopus (17) Google Scholar) resulted in a partial loss of α subunit from the S377D β subunit and a complete loss of α subunit from the S377E β subunit, as described under “Experimental Procedures.” The results suggest that association of the S377D and S377E β subunits with the α subunit is weaker than that of the wild type β subunit, as demonstrated below. The absorption spectra of the pyridoxal phosphate cofactor of the tryptophan synthase β2 subunit and α2β2 complex from E. coli (17Goldberg M.E. Baldwin R.L. Biochemistry. 1967; 6: 2113-2119Crossref PubMed Scopus (60) Google Scholar) and of the α2β2 complex from S. typhimurium (18Peracchi A. Bettati S. Mozzarelli A. Rossi G.L. Miles E.W. Dunn M.F. Biochemistry. 1996; 35: 1872-1880Crossref PubMed Scopus (70) Google Scholar) are pH-independent between pH 6 and 10. The absorption spectra of the S377A β2 subunit and α2β2 complex are also pH-independent (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar). In contrast, the absorption spectra of the S377D β2subunit (Fig.2 A) and of the S377E β2 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. Analysis of plots of absorbance against pH at these wavelengths (Fig.2 B) shows pK a values of 7.63 ± 0.06 for this ionization for the S377D β2 subunit and of 7.89 ± 0.08 for the S377D α2β2complex. The finding that the pK a value for the S377E β2 subunit (7.78; data not shown) is 0.15 pH units higher than that for the S337D β2 subunit (7.63; Fig.2 B) is consistent with the higher pK a value for Glu (4.5) compared with Asp (4.1) in polypeptides and uncharged derivatives of Asp and Glu (19Nozaki Y. Tanford C. J. Biol. Chem. 1967; 242: 4731-4735Abstract Full Text PDF PubMed Google Scholar). The CD spectra of the S377D α2β2 complex show that the ellipticity band with a maximum at 416 nm is also pH-dependent (Fig.2 C). The absorption band centered at 334 nm appears to be optically inactive. The wild type α2β2complex and β2 subunit catalyze β-replacement reactions with l-serine and indole or β-mercaptoethanol that proceed through a series of PLP intermediates (Fig. 1), which have characteristic absorption spectra (20Miles E.W. Dolphin D. Poulson D. Avramovic O. Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects. 1B. John Wiley & Sons, Inc., New York1986: 253-310Google Scholar). Two quinonoid-type intermediates occur in this pathway, the first (E-Q1) after removal of the α-proton ofl-serine and the second (E-Q2) after the β-addition of a nucleophile to the aminoacrylate intermediate (E-AA). E-Q1 has been observed in rapid kinetic studies of the α2β2 complex as a transitory intermediate with maximum absorbance at 460 nm (21Drewe W.J. Dunn M.F. Biochemistry. 1985; 24: 3977-3987Crossref PubMed Scopus (97) Google Scholar), but does not accumulate under equilibrium conditions.E-Q2 accumulates as a stable intermediate under steady-state conditions in reactions of the wild type α2β2 complex with l-serine and nucleophiles, including β-mercaptoethanol and indole (17Goldberg M.E. Baldwin R.L. Biochemistry. 1967; 6: 2113-2119Crossref PubMed Scopus (60) Google Scholar), and with the product, l-tryptophan (22Miles E.W. Hayaishi O. Ishimura Y. Kido R. Biochemical and Medical Aspects of Tryptophan Metabolism. Elsevier/North-Holand Biomedical Press, Amsterdam1980: 137-147Google Scholar, 23Tschopp J. Kirschner K. Biochemistry. 1980; 19: 4514-4521Crossref PubMed Scopus (52) Google Scholar). Mutation of β-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 α2β2complexes (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar). 4The activity of the S377D α2β2 complex is also very low in the reaction with β-chloro-l-alanine and indole and in β-elimination reactions with l-serine or β-chloro-l-alanine. The activities of the S377D α2β2 complexes are rather insensitive to pH between 7 and 9.5 (K.-H. Jhee, unpublished results). Nevertheless, addition of substrates to the S377D α2β2 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 TableI). 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 withl-tryptophan or with l-serine and β-mercaptoethanol. These bands can be attributed toE-Q2 and exhibit absorption maxima at longer wavelengths (Table I) than quinonoid bands observed with the wild type α2β2 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 α2β2 complexes, respectively (Table I), whereas the quinonoid formed from l-serine and β-mercaptoethanol exhibits absorbance maxima at 468 and 508 nm with the wild type and mutant enzymes, respectively. Thel-tryptophan quinonoid has a much greater maximum absorbance with the S377D α2β2 complex (ε504 nm = 49.2 mm−1cm−1) than with the wild type α2β2 complex (ε476 nm = 1.4 mm−1 cm−1) (23Tschopp J. Kirschner K. Biochemistry. 1980; 19: 4514-4521Crossref PubMed Scopus (52) Google Scholar) (Table I).Table ISpectroscopic properties of quinonoid intermediates formed by the S377D α 2 β 2 complexSubstrateFig. 3traceCationλmaxεmaxt maxt 1/2nmmm −1 cm −1minSerK+4981.212.53.1 minSerENH4+4964.117.045 minSerCCs+49817.56.022 minSer + 2-ME1-a2-ME, 2-mercaptoethanol; β-Cl-Ala, β-chloro-l-alanine.Na+5105.009.072 minSer + 2-MEBCs+50838.37.541 minβ-Cl-AlaDCs+49811.86.523 minTrpNa+50447.06.011 hTrpK+50434.24.53.3 hTrpACs+50449.28.024 hTrpNH4+50426.13.57.5 h1-a 2-ME, 2-mercaptoethanol; β-Cl-Ala, β-chloro-l-alanine. Open table in a new tab The quinonoid bands formed differ in stability (Table I). The half-times for disappearance range from 3 min for thel-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 thel-serine intermediate probably reflects either the slow conversion to pyruvate by the very low catalytic activity of the S377D α2β2 complex or the occurrence of irreversible inactivation of the S377D α2β2complex by a reaction intermediate or both (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar). The S377D β2 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 α subunit results in formation of increasing amounts of the quinonoid intermediate. The insetshows a plot of the molar absorbance against the α/β subunit molar ratio. Fit of the data to Equations Equation 3, Equation 4, Equation 5, Equation 6 under “Experimental Procedures” gives values of the apparent dissociation constantK d (αβ) = 7.0 ± 0.2 μm and of 43,000 ± 160 m−1cm−1 for the maximum absorptivity at 504 nm. (The higher value of the maximum absorptivity at 504 nm, 49,200m−1 cm−1, reported in Table I was obtained with more freshly prepared enzyme.) Titration of the S377E β2 subunit with the α subunit by the same method gave values of K d (αβ) = 32 ± 3.6 μm and of 1750 ± 60 m−1cm−1 for the maximum absorptivity. Thus, association of the S377E β subunit with the α subunit is weaker than that of the S377D β subunit, consistent with the greater loss of α subunit during purification of the S377E β subunit from extracts containing α subunit. We cannot directly compare the dissociation constant for the S377D β subunit with that for the wild type β subunit because the constants have not been determined under the same conditions. The apparent dissociation constant for the S377D β subunit in the presence ofl-tryptophan and Cs+(K d (αβ) = 7.0 ± 0.16 μm) is 137-fold higher than the value ofK d (αβ) = 0.051 ± 0.005 μm determined for the wild type α and β subunits from measurements of enzymatic activity in the presence of Cs+in the reaction of l-serine with indole to forml-tryptophan (46Rowlett R. Yang L.-H. Ahmed S.A. McPhie P. Jhee K.-H. Miles E.W. Biochemistry. 1998; 37: 2961-2968Crossref PubMed Scopus (36) Google Scholar) and 3.5-fold higher than the value ofK d (αβ) = 2 μmdetermined from sedimentation equilibrium in the absence of ligands (24Darawshe S. Millar D.B. Ahmed S.A. Miles E.W. Minton A.P. Biophys. Chem. 1997; 69: 53-62Crossref PubMed Scopus (7) Google Scholar). The presence of l-serine is known to tighten the association between the α and β subunits (25Creighton T.E. Yanofsky C. J. Biol. Chem. 1966; 241: 980-990Abstract Full Text PDF PubMed Google Scholar). The weaker association of the S377D β subunit with the α subunit is consistent with loss of approximately one α subunit during purification. The experiments described above were aimed at assessing the functional role of β-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 ε-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 (26Metzler C.M. Cahill A. Metzler D.E. J. Am. Chem. Soc. 1980; 102: 6075-6082Crossref Scopus (250) Google Scholar), 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. Interaction of the carboxylate stabilizes the proton on N-1 (Ha) (see Fig. 5) and increases the pK a by 2–5 units (7Shaw J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (257) Google Scholar, 27Clark P.A. Jansonius J.N. Mehler E.L. J. Am. Chem. Soc. 1993; 115: 9789-9793Crossref Scopus (3) Google Scholar). 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 (27Clark P.A. Jansonius J.N. Mehler E.L. J. Am. Chem. Soc. 1993; 115: 9789-9793Crossref Scopus (3) Google Scholar). Although the electron-accepting properties of the pyridine ring are enhanced by protonation (7Shaw J.P. Petsko G.A. Ringe D. Biochemistry. 1997; 36: 1329-1342Crossref PubMed Scopus (257) Google Scholar), 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 (28Clark P.A. Jansonius J.N. Mehler E.L. J. Am. Chem. Soc. 1993; 115: 1894-1902Crossref Scopus (8) Google Scholar). 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–Ser139hydrogen-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 α2β2 complex shows the presence of no water molecules or other residues that might interact with the carboxylate that has been introduced in the βS377D mutant enzyme. It is possible that the low activities observed for the mutant enzymes (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar)4 result from the absence of residues that modulate the charge density of the introduced carboxylate. Although the pK a 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 (29Kallen R.G. Korpela T. Martell A.E. Matsushima Y. Metzler C.M. Metzler D.E. Morozov Y.V. Ralston I.M. Savin F.A. Torchinsky Y.M. Veno H. Christen P. Metzler D.E. Transaminases. John Wiley & Sons, Inc., New York1985: 60Google Scholar). This result provides evidence that the protonated state of the pyridine nitrogen (N-1) reduces the pK a 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 (pK a = 7.2) from a protonated form (λmax = 420 nm) to a deprotonated form (λmax = 337 nm) in the presence of K+ (30Morino Y. Snell E.E. J. Biol. Chem. 1967; 242: 2800-2809Abstract Full Text PDF PubMed Google Scholar). Aspartate aminotransferase exhibits a pH-dependent conversion (pK a = 6.7) from a protonated internal form (λmax = 430 nm) to a deprotonated form (λmax = 358 nm) (31Yano T. Kuramitsu S. Tanase S. Morino Y. Kagamiyama H. Biochemistry. 1992; 32: 1810-1815Crossref Scopus (46) Google Scholar, 32Yano T. Hinoue Y. Chen V.J. Metzler D.E. Miyahara I. Hirotsu K. Kagamiyama H. J. Mol. Biol. 1993; 234: 1218-1229Crossref PubMed Scopus (34) Google Scholar). 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 pK a of the Schiff base nitrogen (31Yano T. Kuramitsu S. Tanase S. Morino Y. Kagamiyama H. Biochemistry. 1992; 32: 1810-1815Crossref Scopus (46) Google Scholar, 33Onuffer J.J. Kirsch J.F. Protein Eng. 1994; 7: 413-424Crossref PubMed Scopus (61) Google Scholar). Although the spectra of tryptophan synthase are pH-independent (18Peracchi A. Bettati S. Mozzarelli A. Rossi G.L. Miles E.W. Dunn M.F. Biochemistry. 1996; 35: 1872-1880Crossref PubMed Scopus (70) Google Scholar,34Goldberg M.E. York S. Stryer L. Biochemistry. 1968; 7: 3662-3667Crossref PubMed Scopus (76) Google Scholar), the absorption spectra of the mutant enzymes engineered in this work are pH-dependent (Fig. 2). These results provide evidence that the engineered β-Asp377 or β-Glu377 interacts with the protonated N-1 of PLP and lowers the pK a of the imine nitrogen of the internal aldimine, as proposed for aspartate aminotransferase (31Yano T. Kuramitsu S. Tanase S. Morino Y. Kagamiyama H. Biochemistry. 1992; 32: 1810-1815Crossref Scopus (46) Google Scholar, 32Yano T. Hinoue Y. Chen V.J. Metzler D.E. Miyahara I. Hirotsu K. Kagamiyama H. J. Mol. Biol. 1993; 234: 1218-1229Crossref PubMed Scopus (34) Google Scholar). Our finding that the wild type and S377A β2 subunits (11Jhee K.-H. Yang L.-h. Ahmed S.A. McPhie P. Rowlett R. Miles E.W. Protein Sci. 1997; 6: 67Google Scholar) 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 λmax = 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 λmax= 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 λmax = 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 (35Ahmed S.A. McPhie P. Miles E.W. J. Biol. Chem. 1996; 271: 8612-8617Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The enolimine form predominates following a thermally induced reversible conformational transition of the β2subunit (35Ahmed S.A. McPhie P. Miles E.W. J. Biol. Chem. 1996; 271: 8612-8617Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The high pH form of tryptophanase also has λmax = 337 nm, consistent with an enolimine structure (36Metzler C.M. Viswanath R. Metzler D.E. J. Biol. Chem. 1991; 266: 9374-9381Abstract Full Text PDF PubMed Google Scholar, 37June D.S. Suelter C.H. Dye J.L. Biochemistry. 1981; 20: 2707-2713Crossref PubMed Scopus (23) Google Scholar, 38June D.S. Suelter C.H. Dye J.L. Biochemistry. 1981; 20: 2714-2719Crossref PubMed Scopus (26) Google Scholar). The dipolar ionic form (structure IIb) is the structure that has been suggested for the high pH form of aspartate aminotransferase, which has λmax = 360 nm (39Goldberg J.M. Swanson R.V. Goodman H.S. Kirsch J.F. Biochemistry. 1991; 30: 305-312Crossref PubMed Scopus (83) Google Scholar). 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 (40Metzler D.E. Methods Enzymol. 1997; 280: 30-40Crossref PubMed Scopus (1) Google Scholar). Hb dissociates around a pK a value of 6.4 with aspartate aminotransferase (40Metzler D.E. Methods Enzymol. 1997; 280: 30-40Crossref PubMed Scopus (1) Google Scholar). 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 α-carbon into the pyridine ring (1Metzler D.E. Ikawa M. Snell E.E. J. Am. Chem. Soc. 1954; 76: 648-652Crossref Scopus (471) Google Scholar). 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-α–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 andN-methylpyridoxal (41Metzler C.M. Harris A.G. Metzler D.E. Biochemistry. 1988; 27: 4923-4933Crossref PubMed Scopus (34) Google Scholar). 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′ (41Metzler C.M. Harris A.G. Metzler D.E. Biochemistry. 1988; 27: 4923-4933Crossref PubMed Scopus (34) Google Scholar,42Chen V.J. Metzler D.E. Jenkins W.T. J. Biol. Chem. 1987; 262: 14422-14427Abstract Full Text PDF PubMed Google Scholar), as shown in the structure in Fig. 3. We conclude that replacing tryptophan synthase β 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.