Portal de Periódicos da CAPES

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function, and Stability

2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês

10.1074/jbc.m305138200

1083-351X

Barbara Campanini, Samanta Raboni, Simona Vaccari, Lei Zhang, Paul Cook, Theodore L. Hazlett, Andrea Mozzarelli, Stefano Bettati,

Nitrogen and Sulfur Effects on Brassica

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5′-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions. O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5′-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions. The biosynthesis of cysteine in bacteria and plants is accomplished by the pyridoxal 5′-phosphate (PLP) 1The abbreviations used are: PLP, pyridoxal 5′-phosphate; OASS, O-acetylserine sulfhydrylase; OAS, O-acetyl-l-serine; TNB, 5-thio-2-nitrobenzoate; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; GdnHCl, guanidine hydrochloride; SAT, serine acetyltransferase; WT, wild type.-dependent enzyme O-acetylserine sulfhydrylase (OASS). PLP-dependent enzymes are currently classified into three functional families, depending on the mechanism of the catalyzed reaction, and into 5-fold types, depending on the structural arrangement. OASS belongs to the β-family and to the fold type II, with the overall reaction catalyzed by the enzyme being a β-replacement. Members of the β-family include, among others, the β-subunit of tryptophan synthase, cystathionine β-synthase, and threonine dehydratase. Depending on the subcellular compartment and on the growth conditions, many isoforms of OASS have been described (1Kuske C.R. Hill K.K. Guzman E. Jackson P.J. Plant Physiol. 1996; 112: 659-667Crossref PubMed Scopus (32) Google Scholar, 2Tai C.H. Nalabolu S.R. Jacobson T.M. Minter D.E. Cook P.F. Biochemistry. 1993; 32: 6433-6442Crossref PubMed Scopus (110) Google Scholar). In Salmonella typhimurium, the OASS-A isoform is responsible for the synthesis of l-cysteine from sulfide and O-acetylserine (OAS). Catalysis follows a Bi Bi ping-pong kinetic mechanism (2Tai C.H. Nalabolu S.R. Jacobson T.M. Minter D.E. Cook P.F. Biochemistry. 1993; 32: 6433-6442Crossref PubMed Scopus (110) Google Scholar, 3Cook P.F. Wedding R.T. J. Biol. Chem. 1976; 251: 2023-2029Abstract Full Text PDF PubMed Google Scholar) and is accompanied by large conformational changes that result in the transition from an open to a closed form of the enzyme (4Burkhard P. Tai C.H. Ristroph C.M. Cook P.F. Jansonius J.N. J. Mol. Biol. 1999; 291: 941-953Crossref PubMed Scopus (120) Google Scholar, 5McClure G.D. Cook P.F. Biochemistry. 1994; 33: 1674-1683Crossref PubMed Scopus (59) Google Scholar). The conformational changes are triggered by the hydrogen bonding of the α-carboxylate group of the substrate OAS to Asn69 and to other residues belonging to the "asparagine loop." The rearrangement of the side chain of Asn69 is transmitted to a subdomain of the N-terminal domain that rotates by 13° from the position occupied in the native enzyme. The conformational changes result in the formation of new hydrogen bonds and hydrophobic interactions between the N-and C-terminal domains. The microenvironment generated by these structural rearrangements stabilizes and orients the external aldimine for the elimination reaction and protects the highly reactive α-aminoacrylate intermediate formed upon acetate elimination. A low thermodynamic stability was evidenced for holo-OASS (6Bettati S. Benci S. Campanini B. Raboni S. Chirico G. Beretta S. Schnackerz K.D. Hazlett T.L. Gratton E. Mozzarelli A. J. Biol. Chem. 2000; 275: 40244-40251Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Most of the stabilization free energy was demonstrated to derive from the binding of the cofactor to the active site, with a more pronounced effect on the C-terminal domain (7Bettati S. Campanini B. Vaccari S. Mozzarelli A. Schianchi G. Hazlett T.L. Gratton E. Benci S. Biochim. Biophys. Acta. 2002; 1596: 47-54Crossref PubMed Scopus (9) Google Scholar), which appears to be more stable than the N-terminal domain. The lower stability of the N-terminal domain supports the notion of a relationship between a marginal structural stabilization and the flexibility required for catalysis. OASS possesses two tryptophan residues: Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Both residues are exposed to solvent, with Trp161 located in a more hydrophilic environment than Trp50 (Fig. 1) (8Burkhard P. Rao G.S.J. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (180) Google Scholar). Upon excitation at 298 nm, an energy transfer process takes place between the tryptophans and PLP. Structural data (8Burkhard P. Rao G.S.J. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (180) Google Scholar) indicate that, although both tryptophans are at the right distance from PLP to transfer their excitation energy to the cofactor, only Trp50 is correctly oriented for an efficient process to take place. Tryptophan residues are often well conserved in protein sequences (9Uchiyama T. Katouno F. Nikaidou N. Nonaka T. Sugiyama J. Watanabe T. J. Biol. Chem. 2001; 276: 41343-41349Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 10Greene L.H. Chrysina E.D. Irons L.I. Papageorgiou A.C. Acharya K.R. Brew K. Protein Sci. 2001; 10: 2301-2316Crossref PubMed Scopus (67) Google Scholar, 11Hillier B.J. Rodriguez H.M. Gregoret L.M. Fold Des. 1998; 3: 87-93Abstract Full Text Full Text PDF PubMed Google Scholar, 12Eason D.D. Shepherd A.T. Blanck G. Biochim. Biophys. Acta. 1999; 1446: 140-144Crossref PubMed Scopus (13) Google Scholar) and are associated with a wide variety of protein functions, from ligand binding (9Uchiyama T. Katouno F. Nikaidou N. Nonaka T. Sugiyama J. Watanabe T. J. Biol. Chem. 2001; 276: 41343-41349Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Bray M.R. Johnson P.E. Gilkes N.R. McIntosh L.P. Kilburn D.G. Warren R.A. Protein Sci. 1996; 5: 2311-2318Crossref PubMed Scopus (53) Google Scholar, 14Fulton K.F. Jackson S.E. Buckle A.M. Biochemistry. 2003; 42: 2364-2372Crossref PubMed Scopus (34) Google Scholar) to DNA-protein interactions (12Eason D.D. Shepherd A.T. Blanck G. Biochim. Biophys. Acta. 1999; 1446: 140-144Crossref PubMed Scopus (13) Google Scholar, 15Marie G. Serani L. Laprevote O. Cahuzac B. Guittet E. Felenbok B. Protein Sci. 2001; 10: 99-107Crossref PubMed Scopus (9) Google Scholar, 16Zargarian L. Le Tilly V. Jamin N. Chaffotte A. Gabrielsen O.S. Toma F. Alpert B. Biochemistry. 1999; 38: 1921-1929Crossref PubMed Scopus (26) Google Scholar) and from structure stabilization (10Greene L.H. Chrysina E.D. Irons L.I. Papageorgiou A.C. Acharya K.R. Brew K. Protein Sci. 2001; 10: 2301-2316Crossref PubMed Scopus (67) Google Scholar, 17Subramaniam V. Jovin T.M. Rivera-Pomar R.V. J. Biol. Chem. 2001; 276: 21506-21511Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 18Davidson W.S. Arnvig-McGuire K. Kennedy A. Kosman J. Hazlett T.L. Jonas A. Biochemistry. 1999; 38: 14387-14395Crossref PubMed Scopus (69) Google Scholar) to protein-protein interactions (19Wallace L.A. Burke J. Dirr H.W. Biochim. Biophys. Acta. 2000; 1478: 325-332Crossref PubMed Scopus (21) Google Scholar, 20Freitag S. Le Trong I. Chilkoti A. Klumb L.A. Stayton P.S. Stenkamp R.E. J. Mol. Biol. 1998; 279: 211-221Crossref PubMed Scopus (73) Google Scholar). Recently, it has been observed that often tryptophan residues are preferentially located in stable regions of proteins (21Wrabl J.O. Larson S.A. Hilser V.J. Protein Sci. 2001; 10: 1032-1045Crossref PubMed Scopus (25) Google Scholar), and their substitution with other amino acids commonly results in the destabilization of the structure and in the modification of the folding/unfolding mechanism (see Ref. 14Fulton K.F. Jackson S.E. Buckle A.M. Biochemistry. 2003; 42: 2364-2372Crossref PubMed Scopus (34) Google Scholar and references therein) (22Honda Y. Fukamizo T. Okajima T. Goto S. Boucher I. Brzezinski R. Biochim. Biophys. Acta. 1999; 1429: 365-376Crossref PubMed Scopus (19) Google Scholar, 23Watanabe H. Kragh-Hansen U. Tanase S. Nakajou K. Mitarai M. Iwao Y. Maruyama T. Otagiri M. Biochem. J. 2001; 357: 269-274Crossref PubMed Google Scholar). In this work, we prepared two single tryptophan mutants of OASS: W50Y and W161Y. The presence of a single tryptophan residue in the mutants allows separate probes of the structure and dynamics of the N- and C-terminal domains. The aim of the present work was to characterize the role of the tryptophans in the structure, function, and stability of OASS and to gain insight into the unfolding processes of the N- and C-terminal domains. Chemicals and Molecular Biology Reagents—Hepes, O-acetyl-l-serine, 5,5′-dithiobis(2-nitrobenzoate), PLP, LB broth, citric acid, magnesium chloride, KH2PO4, Na(NH4)HPO4, thiamine, l-tryptophan, reduced glutathione, l-leucine, ampicillin, and streptomycin sulfate were from Sigma. Dithiothreitol and guanidine hydrochloride were from Fluka, and p-terphenyl was from Aldrich. Restriction enzymes and DNA ligase were from Amersham Biosciences, Promega, or U.S. Biochemical Corp. Oligonucleotides used for mutagenesis and sequencing were from Invitrogen or MWG-Biotech AG. All of the reagents were of the best commercially available quality and were used without further purification. Bacterial Strains and Expression Vectors—The single tryptophan mutants were expressed in Escherichia coli NK3 cells that lack cysK and cysM genes. The cysK gene encodes for OASS-A, whereas cysM encodes for OASS-B (24Tai C.H. Cook P.F. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 185-234PubMed Google Scholar). The plasmid pRSM40 (25Rege V.D. Kredich N.M. Tai C.H. Karsten W.E. Schnackerz K.D. Cook P.F. Biochemistry. 1996; 35: 13485-13493Crossref PubMed Scopus (39) Google Scholar) was used as the expression vector for the W50Y mutant. The plasmid pCKM3, used for the expression of the W161Y mutant, contains the cysK gene and its natural promoter on an EcoRI/SphI fragment from pRSM40. The expression of S. typhimurium OASS-A in both pRSM40 and pCKM3 constructs is under the control of the natural promoter for cysK. Site-directed Mutagenesis—The mutation Trp50 → Tyr was introduced using a PCR-based method with the following forward and reverse primers: 5′-GCCGTATCGGCGCCAACATGATTTATGATGCCGAAAAGCG-3′ and 5′-GCAGCAACCGCGGCGCCGGAAG-3′. The sequence coding for the tyrosine residue is indicated in boldface type, and the restriction sites for the enzyme NarI are underlined. WT pRSM40 was digested with NarI and purified from agarose gel to generate the vector. The amplified fragment from the PCR carrying the mutation TGG → TAT was digested with NarI, and the insert was purified from agarose gel and cloned into the vector. The W161Y mutant was obtained using the kit Altered Sites® II (Promega). The mutation was introduced using the following primer: 5′-GGCCCGGAAATCTATGAAGACACCGAT-3′. The EcoRI/SphI fragment from pRSM40 was cloned into the mutagenesis vector pALTER. E. coli strain ES1301 was used for plasmid propagation and plasmid isolation. E. coli strain JM109 was used for plasmid long term maintenance. Following mutagenesis, the EcoRI/SphI fragment carrying the mutation was cloned into pBR322 to generate the expression vector pCKM3. Single Tryptophan Mutants Expression and Purification—Cultures of E. coli NK3 transformed with mutagenized pCKM3 or pRSM40 were grown at 37 °C in a fermentor using a medium composed of Vogel Bonner E supplemented with 0.5% glucose, 5% LB, 50 μm thiamine, 40 μml-tryptophan, 0.5 mm reduced glutathione, 50 mm l-leucine, and 100 μg/ml ampicillin. The cells were harvested by centrifugation, and the protein was partially purified by streptomycin and ammonium sulfate precipitation as described previously (26Hara S. Payne M.A. Schnackerz K.D. Cook P.F. Protein Expression Purif. 1990; 1: 70-76Crossref PubMed Scopus (38) Google Scholar). The chromatographic procedure for the purification of the W50Y mutant was similar to that of the WT enzyme (2Tai C.H. Nalabolu S.R. Jacobson T.M. Minter D.E. Cook P.F. Biochemistry. 1993; 32: 6433-6442Crossref PubMed Scopus (110) Google Scholar) and was followed by a final size exclusion chromatographic step on an Ultrogel-AcA 44 column (IBF Biotechnics). The purification of the W161Y mutant was carried out through a three-step chromatographic procedure on a HiTrap DEAE FF column (Amersham Biosciences), an Ultrogel-AcA 44 column (IBF Biotechnics), and a HiTrap Q Sepharose HP column (Amersham Biosciences). Based on SDS-PAGE, the W50Y and W161Y mutants were 99% and 90–95% pure, respectively. Buffers—Absorbance, steady-state, and time-resolved fluorescence experiments were carried out in a buffer solution containing 100 mm Hepes, pH 7.0, in the absence and presence of defined concentrations of GdnHCl. Circular dichroism measurements were carried out in buffer solutions containing 20 mm potassium phosphate, pH 7.0, and different concentrations of GdnHCl. Denaturant-containing solutions were prepared according to Pace (27Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure. 2nd Ed. Oxford University Press, Oxford, UK1997Google Scholar). GdnHCl concentration was determined by measuring the solution refractive index. Absorption, Steady-state Fluorescence, and Circular Dichroism Measurements—Absorption measurements were carried out using a Cary 400 Scan or, for the activity assays, a Cary 219 spectrophotometer. Fluorescence spectra were collected on either a PerkinElmer Life Sciences LS50B or a SPEX Fluoromax-2 photon-counting fluorometer (Jobin-Yvon). Circular dichroism measurements were carried out using a JASCO J-715 spectropolarimeter. Each spectrum is the average of three measurements. Fractions of α and β structure were calculated by deconvoluting the spectra with CD Spectra Deconvolution software, version 2.1 (Gerald Böhm; see Ref. 28Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 5: 191-195Crossref PubMed Scopus (1015) Google Scholar). Spectra were acquired at 20 ± 0.5 °C and were corrected for solvent contribution. Spectra of the single tryptophan mutants, equilibrated at different GdnHCl concentrations, were acquired after an incubation of 24 h at 20 °C. Fluorescence Quenching Measurements—The accessibility of the cofactor of WT and W161Y OASS was assessed by fluorescence quenching with acrylamide. Acrylamide was chosen due to its neutral nature and the absence of ionic strength effects on the fluorescence properties of the cofactor. Experiments on the WT protein were performed both for the open and the closed form of the enzyme. Experiments were carried out on solutions containing 40 μm OASS and either 100 mm Hepes, pH 7.0, for the open form of the enzyme or 100 mm Ches, pH 9.0, in the presence of 100 mm l-Ser for the closed form. Spectra, collected upon excitation at 330 nm, were corrected for solvent contribution. Data were analyzed according to a modified Stern-Volmer equation (29Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1625) Google Scholar), assuming two fluorescent and noninteracting species, where only species A is quenchable by acrylamide, F0F=1+KSVA[Q](1+KSVA[Q])(1-fA)+fA(Eq. 1) where F 0 is the fluorescence intensity in the absence of the quencher, F is the fluorescence at each given quencher concentration, KSVA is the Stern-Volmer constant relative to the accessible site A, [Q] is the concentration of the quencher, and fA is the fraction of initial fluorescence emitted by species A. Time-resolved Fluorescence Measurements—Fluorescence intensity decays were measured by the phase and modulation technique (30Spencer R.D. Weber G. Ann. N. Y. Acad. Sci. 1969; 158: 361-376Crossref Scopus (531) Google Scholar, 31Gratton E. Limkeman M. Biophys. J. 1983; 44: 315-324Abstract Full Text PDF PubMed Scopus (417) Google Scholar). The instrument set up was described previously (7Bettati S. Campanini B. Vaccari S. Mozzarelli A. Schianchi G. Hazlett T.L. Gratton E. Benci S. Biochim. Biophys. Acta. 2002; 1596: 47-54Crossref PubMed Scopus (9) Google Scholar). Tryptophan fluorescence lifetimes of the single tryptophan mutants were measured at a protomer concentration of 3.3 μm, upon excitation at 295 nm. A p-terphenyl solution in ethanol (1.05 ns) was used as a lifetime standard reference. To eliminate polarization artifacts in the intensity decay, data were collected under magic angle conditions with the excitation light polarized normal to the laboratory plane, 0°, and the emission polarizer oriented at 54.7° (30Spencer R.D. Weber G. Ann. N. Y. Acad. Sci. 1969; 158: 361-376Crossref Scopus (531) Google Scholar). Samples were equilibrated at 20 ± 0.5 °C using a jacketed cell holder and a circulating water bath. Data were fitted to a sum of discrete exponentials (32Jameson D.M. Hazlett T.L. Dewey T.G. Biophysical and Biochemical Aspects of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1991: 106-133Google Scholar) with lifetime τ i and fractional intensity fi by the Marquardt algorithm of the Globals Unlimited software (University of Illinois, Urbana, IL) (33Beechem J.M. Gratton E. Lakowicz J.R. Time-Resolved Laser Spectroscopy in Biochemistry, SPIE Proceedings Vol. 909. International Society for Optical Engineering, Bellingham, WA1988: 70-81Google Scholar). A short component of 1 ps was introduced to account for any Rayleigh or Raman scattering contributions (32Jameson D.M. Hazlett T.L. Dewey T.G. Biophysical and Biochemical Aspects of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1991: 106-133Google Scholar). The fractional contribution of the short component to the total emission was always less than 3%. Frequency-independent S.E. values of 0.2° for phase data and 0.004 for modulation data were routinely applied. The χ2 minimization was the criterion used to select the best fits (31Gratton E. Limkeman M. Biophys. J. 1983; 44: 315-324Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 32Jameson D.M. Hazlett T.L. Dewey T.G. Biophysical and Biochemical Aspects of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1991: 106-133Google Scholar). Equilibrium Denaturation Curves—The dependence of signal intensity I on denaturant concentration [D] was fitted to a two-state model according to the equation, I=IN+(IU+SU[D])e([D]-D50)mRT1+e([D]-D50)mRT(Eq. 2) where I is either the fluorescence emission intensity at a given wavelength or the mean residue ellipticity at 222 nm, IN and IU are the extrapolated signal values for the native and denatured protein, respectively, SU is a post-transitional slope reflecting the dependence of the signal on denaturant concentration, D 50 is the denaturant concentration at half-transition, and m is the denaturant index. The experimental data points were converted to fraction of total effect FU by using the fitting parameters previously determined, according to the equation, FU=IN-IIN-(IU+SU[D])(Eq. 3) The equilibrium unfolding constants KU at each denaturant concentration were calculated from the equation, KU=FU1-FU(Eq. 4) The KU values were used to calculate the unfolding free energies. ΔGU0=-RTlnKU(Eq. 5) ΔG0,U0, the free energy change in the absence of denaturant, and m were calculated using the linear extrapolation method (34Greene R.F. Pace C.N. J. Biol. Chem. 1974; 249: 5388-5393Abstract Full Text PDF PubMed Google Scholar). ΔGU0=ΔG0,U0-m[D](Eq. 6) D 50 was calculated as follows: D50=ΔG0,U0m(Eq. 7) Reversibility of the Denaturation Reaction—The calculation of thermodynamic parameters from equilibrium denaturation curves requires that the denaturation reaction is fully reversible (27Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure. 2nd Ed. Oxford University Press, Oxford, UK1997Google Scholar). The reversibility of the denaturation of WT holo-OASS and of W161Y and W50Y mutants was assessed diluting a solution of the unfolded enzyme in 100 mm Hepes, pH 7.0, and monitoring the kinetics of the refolding reaction via fluorescence emission spectroscopy upon excitation at 298 nm. The native emission spectrum of WT holo-OASS was recovered within 1 h. A similar result was obtained for W161Y mutant. On the contrary, the reversibility of the unfolding of the W50Y mutant appears to be only partial, since the enzyme never recovers the fluorescence emission spectrum typical of the native protein. For this reason, the fitting of the equilibrium denaturation curves of W50Y mutant only provides indicative D 50 values, useful for comparison with the WT enzyme but devoid of any thermodynamic significance. Enzyme Activity—A schematic mechanism for the OASS-A reaction is given in Scheme 1. where A, B, and P represent OAS, TNB, and acetate, respectively; E is the free enzyme, E(SB1) is the OAS external Schiff base, E(SB2) is the S-(3-carboxy-4-nitrophenyl)-l-cysteine external Schiff base, and E(AA) is the α-aminoacrylate Schiff base. For the reported kinetic mechanism, when k 5 is fast compared with k 3 and k 4 (see also Refs. 35Hwang C.C. Woehl E.U. Minter D.E. Dunn M.F. Cook P.F. Biochemistry. 1996; 35: 6358-6365Crossref PubMed Scopus (29) Google Scholar and 36Woehl E.U. Tai C.H. Dunn M.F. Cook P.F. Biochemistry. 1996; 35: 4776-4783Crossref PubMed Scopus (58) Google Scholar), the following expressions for k cat, K OAS, and K TNB apply, VEt=k31+k3k91+k10k11+k3k11(Eq. 8) KOAS=Kd(OAS)1+k3k21+k31+k10k11k9+k3k11(Eq. 9) where Kd (OAS) = k 2/k 1, and KTNB=Kd(TNB)k9k8+1+k10k11k91k3+1k11+1+k10k11(Eq. 10) where Kd (TNB) = k 8/k 7. Enzyme activity was assayed using OAS and TNB as substrates in 100 mm Hepes, pH 7.0, at 20 °C (2Tai C.H. Nalabolu S.R. Jacobson T.M. Minter D.E. Cook P.F. Biochemistry. 1993; 32: 6433-6442Crossref PubMed Scopus (110) Google Scholar). OAS and TNB concentrations were varied over a range of 0.5–2 mm and 25–100 μm, respectively. Since under these conditions neither substrate inhibits OASS activity, the initial velocity patterns were fitted to the equation for a simple Bi Bi ping-pong kinetic mechanism (37Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, Inc., New York1993Google Scholar) υ=Vmax[OAS][TNB]KOAS[TNB]+KTNB[OAS]+[OAS][TNB](Eq. 11) Sequence Alignments—Sequences of procaryotic and eucaryotic OASS were retrieved from a nonredundant sequence data base using the BlastP program (38Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70715) Google Scholar). Sequences were aligned using ClustalW (39Higgins D.G. Sharp P.M. Gene (Amst.). 1988; 73: 237-244Crossref PubMed Scopus (2878) Google Scholar, 40Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55752) Google Scholar) with default parameters. Absorbance Spectra of W50Y and W161Y Mutants—The UV-visible spectra of the single tryptophan mutants are qualitatively similar to the spectrum of the WT protein, showing two peaks centered at 278 and 412 nm (Fig. 2). Both mutations cause a decrease of the intensity of the peak at 278 nm, due to the lower extinction coefficient of the tyrosine residue with respect to tryptophan. The peak at 412 nm, attributed to the ketoenamine tautomer of the PLP internal aldimine (3Cook P.F. Wedding R.T. J. Biol. Chem. 1976; 251: 2023-2029Abstract Full Text PDF PubMed Google Scholar, 41Becker M.A. Kredich N.M. Tomkins G.M. J. Biol. Chem. 1969; 244: 2418-2427Abstract Full Text PDF PubMed Google Scholar, 42Cook P.F. Hara S. Nalabolu S. Schnackerz K.D. Biochemistry. 1992; 31: 2298-2303Crossref PubMed Scopus (62) Google Scholar), is still present, indicating that the mutation has not hampered the binding of PLP to the protein active site. The ratio between the intensity at 280 nm and at 412 nm is 3.4–3.6 in WT OASS and 2.8 and 2.6 in W161Y and W50Y mutants, respectively. The spectrum of the W161Y mutant shows a slightly higher absorbance at 330 nm with respect to the WT OASS and to the W50Y mutant, suggesting the presence of an increased population of the enolimine tautomer of the internal aldimine (43Faeder E.J. Hammes G.G. Biochemistry. 1971; 10: 1041-1045Crossref PubMed Scopus (62) Google Scholar). Fluorescence Emission Spectra of Single Tryptophan Mutants—Emission spectra of the single tryptophan mutants were collected upon excitation at 298 nm (Fig. 3). The spectra are characterized by a major peak, centered at about 335 nm, due to the direct tryptophan emission, and a minor peak centered at 500 nm that, in the WT enzyme, was attributed to the energy transfer process occurring predominantly between Trp50 and the cofactor (8Burkhard P. Rao G.S.J. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (180) Google Scholar, 44Strambini G.B. Cioni P. Cook P.F. Biochemistry. 1996; 35: 8392-8400Crossref PubMed Scopus (39) Google Scholar). In the WT enzyme, the ratio between the tryptophans and the cofactor emissions is about 7. The peak intensity at 500 nm for the W161Y mutant and the WT protein is the same, thus reinforcing the concept of an energy transfer process that involves Trp50 as the principal donor. Unexpectedly, also the W50Y mutant shows a band at 500 nm, which exhibits an intensity about half of that of the wild type protein, indicating the occurrence of an energy transfer process between Trp161 and PLP. The ratio between the emission at 330 nm and the emission at 500 nm for the W161Y mutant is about 2.6, whereas the ratio increases to 5.7 for the W50Y mutant. The sum of the spectra of the two mutants gives a spectrum that is not superimposable with that of the WT enzyme (Fig. 3). In particular, the tryptophan emission intensity is lower, and the cofactor emission is about twice that measured for the wild type protein. Fluorescence Lifetimes of W50Y and W161Y Mutants and Comparison with the WT Holo-OASS—The effect of the mutations on the local environment of the remaining tryptophan residue was examined by measuring the fluorescence emission lifetimes of the native protein (Table I). Fluorescence lifetimes are very sensitive to tryptophan microenvironment, allowing the detection of small changes in their solvent exposure. The fluorescence emission decay of WT holo-OASS is well described by three discrete species (7Bettati S. Campanini B. Vaccari S. Mozzarelli A. Schianchi G. Hazlett T.L. Gratton E. Benci S. Biochim. Biophys. Acta. 2002; 1596: 47-54Crossref PubMed Scopus (9) Google Scholar). The longer lifetime (5.5 ns) was mainly attributed to Trp50 emission, whereas the intermediate (2.1 ns) lifetime was mainly attributed to Trp161 emission (Table I) (7Bettati S. Campanini B. Vaccari S. Mozzarelli A. Schianchi G. Hazlett T.L. Gratton E. Benci S. Biochim. Biophys. Acta. 2002; 1596: 47-54Crossref PubMed Scopus (9) Google Scholar). Fluorescence emission decays of single tryptophan mutants were fitted to a sum of two exponentials plus a fixed short component (1 ps), accounting for the scattered light (Table I). The W50Y mutant is characterized by a lifetime of 2.3 ns, accounting for 91% of the total emission, and a short lifetime of about 0.8 ns. The W161Y mutant fluorescence decay is dominated by a short lifetime (0.9 ns), accounting for 75% of the emission, accompanied by a longer lifetime (4.7 ns), accounting for 25% of the emission. This finding confirms the lifetime attribution carried out on the WT enzyme (7Bettati S. Campanini B. Vaccari S. Mozzarelli A. Schianchi G. Hazlett T.L. Gratton E. Benci S. Biochim. Biophys. Acta. 2002; 1596: 47-54Crossref PubMed Scopus (9) Google Scholar) and indicates that the substitution of the tryptophan residue in one domain has no significant long range effects on the microenvironment of the tryptophan in the other domain.Table IFluorescence lifetimes and fractional intensities for WT OASS, W50Y mutant, and W161Y mutantOASSτ1τ2τ3f1f2f3nsnsnsWT5.5 (Trp50)