FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061178897> ?p ?o ?g. }

• W2061178897 endingPage "1325" @default.
• W2061178897 startingPage "1320" @default.
• W2061178897 abstract "Tyrosine phenol-lyase (TPL), which catalyzes the β-elimination reaction of l-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the α-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from l-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the β-elimination activity toward dicarboxylic amino acids at least 104-fold. Dicarboxylic amino acids (l-aspartate,l-glutamate, and l-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids,e.g. formate in the case of l-aspartate. The activity toward l-aspartate (k cat = 0.21 s−1) was two times higher than that towardl-tyrosine. β-Elimination and transamination as a minor side reaction (k cat = 0.001 s−1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid β-lyase is an enzyme not found in nature. Tyrosine phenol-lyase (TPL), which catalyzes the β-elimination reaction of l-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the α-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from l-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the β-elimination activity toward dicarboxylic amino acids at least 104-fold. Dicarboxylic amino acids (l-aspartate,l-glutamate, and l-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids,e.g. formate in the case of l-aspartate. The activity toward l-aspartate (k cat = 0.21 s−1) was two times higher than that towardl-tyrosine. β-Elimination and transamination as a minor side reaction (k cat = 0.001 s−1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid β-lyase is an enzyme not found in nature. The pyridoxal 5′-phosphate-dependent enzymes (B6 enzymes) catalyze a wide variety of reactions in the metabolism of amino acids (1John R.A. Biochim. Biophys. Acta. 1995; 1248: 81-96Crossref PubMed Scopus (326) Google Scholar). A comparison of amino acid sequences has shown that the majority of B6 enzymes belong to the large α/γ-superfamily of homologous B6 enzymes (2Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (343) Google Scholar, 3Huber B. Evolutionary Relationships among Pyridoxal 5′-phosphate-dependent Enzymes. Diploma thesis, University of Zurich, 1996Google Scholar). Tyrosine phenol-lyase (TPL) 1The abbreviations used are: TPL, tyrosine phenol-lyase; AspAT, aspartate aminotransferase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate. 1The abbreviations used are: TPL, tyrosine phenol-lyase; AspAT, aspartate aminotransferase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate. of Citrobacter freundii is a member of the α-family. It catalyzes the β-elimination of l-tyrosine to produce phenol, pyruvate, and ammonium (Equation 1). L­Tyrosine+H2O⇌phenol+pyruvate+NH4+Equation 1 A number of amino acids with suitable leaving groups on Cβ, such as l-serine and O-acyl-l-serines (4Phillips R.S. Arch. Biochem. Biophys. 1987; 256: 302-310Crossref PubMed Scopus (47) Google Scholar), l-cysteine, S-alkyl-l-cysteines (4Phillips R.S. Arch. Biochem. Biophys. 1987; 256: 302-310Crossref PubMed Scopus (47) Google Scholar, 5Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar), and 3-chloro-l-alanine, are also substrates for β-elimination. Moreover, TPL has been found to catalyze markedly slower side reactions, i.e. β-replacement reactions (6Kumagai H. Matsui H. Ohkishi H. Ogata K. Yamada H. Ueno T. Fukami H. Biochem. Biophys. Res. Commun. 1969; 34: 266-270Crossref PubMed Scopus (62) Google Scholar,7Ueno T. Fukami H. Ohkishi H. Kumagai H. Yamada H. Biochim. Biophys. Acta. 1970; 206: 476-479Crossref PubMed Scopus (36) Google Scholar), racemization of alanine (8Kumagai H. Kashima N. Yamada H. Biochem. Biophys. Res. Commun. 1970; 39: 796-801Crossref PubMed Scopus (56) Google Scholar, 9Chen H. Phillips R.S. Biochemistry. 1993; 32: 11591-11599Crossref PubMed Scopus (38) Google Scholar), as well as transamination reactions of its substrates l-tyrosine, l-serine, and of the competitive inhibitors l-alanine,l-phenylalanine, and l-m-tyrosine (10Demidkina T.V. Myagkikh I.V. Azhayev A.V. Eur. J. Biochem. 1987; 170: 311-316Crossref PubMed Scopus (18) Google Scholar). X-ray crystallographic structure analysis has shown the folding pattern of the polypeptide chain of tetrameric TPL from C. freundiito be similar to that of dimeric aspartate aminotransferase (AspAT) (11Antson A.A. Strokopytov B.V. Murshudov G.N. Isupov M.N. Harutyunyan E.H. Demidkina T.V. Vassyiyer D.G. Dauter Z. Terry H. Wilson K.S. FEBS Lett. 1992; 302: 256-260Crossref PubMed Scopus (13) Google Scholar), which, like TPL, is a member of the α-family of pyridoxal 5′-phosphate (PLP)-dependent enzymes. Despite their similarity in secondary and tertiary structure, the two enzymes show only low sequence identity, e.g. 23% between TPL ofC. freundii and AspAT of Escherichia coli. AspAT catalyzes the reversible transamination reaction of the dicarboxylicl-amino acids aspartate and glutamate with the cognate 2-oxo acids 2-oxoglutarate and oxalacetate. The structures of the active sites of TPL and AspAT are similar; most of the residues that participate in the binding of the coenzyme and the α-carboxylate group of the substrate in AspAT (12Kirsch 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 (420) Google Scholar) are conserved in the structure of TPL (13Antson 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 (130) Google Scholar). Obviously, these two homologous enzymes use the same protein scaffold to catalyze different reactions with different substrates. Thus, alteration of the specificity of a given enzyme by substitution of a limited number of critical amino acid residues seems feasible. Alignments of amino acid sequences of homologous enzymes may be used to identify the residues underlying the differences in their reaction and substrate specificity. Substitution of the residues to which the substrate binds has proven successful in changing the substrate specificity of several enzymes without destroying their catalytic apparatus (14Wilks H.M. Hart K.W. Feeney R. Dunn C.R. Muirhead H. Chia W.N. Barstow D.A. Atkinson T. Clarke A.R. Holbrook J.J. Science. 1988; 242: 1541-1544Crossref PubMed Scopus (243) Google Scholar, 15Bone R. Silen J.L. Agard D.A. Nature. 1989; 339: 191-195Crossref PubMed Scopus (155) Google Scholar, 16Henderson G.B. Murgolo N.J. Kuriyan J. Osapay K. Kominos D. Berry A. Scrutton N.S. Hinchliffe N.W. Perham R.N. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8769-8773Crossref PubMed Scopus (51) Google Scholar, 17Khouri H.E. Vernet T. Menard R. Parlati F. Laflamme P. Tessier D.C. Gour-Salin B. Thomas D.Y. Storer A.C. Biochemistry. 1991; 30: 8929-8936Crossref PubMed Scopus (85) Google Scholar, 18Green D.W. Sun H.-W. Plapp B.V. J. Biol. Chem. 1993; 268: 7792-7798Abstract Full Text PDF PubMed Google Scholar, 19Onuffer J.J. Kirsch J.F. Protein Sci. 1995; 4: 1750-1757Crossref PubMed Scopus (71) Google Scholar, 20Ballinger M.D. Tom J. Wells J.A. Biochemistry. 1995; 34: 13312-13319Crossref PubMed Scopus (73) Google Scholar, 21Ballinger M.D. Tom J. Wells J.A. Biochemistry. 1996; 35: 13579-13585Crossref PubMed Scopus (59) Google Scholar). This paper reports a homology modeling approach that, together with information obtained from structural and mechanistic studies of AspAT, was used to redesign the substrate specificity of TPL in favor of dicarboxylic amino acids. Plasmid pTZTPL (22Chen H. Gollnick P. Phillips R.S. Eur. J. Biochem. 1995; 229: 540-549Crossref PubMed Scopus (46) Google Scholar) containing the entire coding sequence of C. freundiityrosine phenol-lyase was used as template for in vitromutagenesis. The tpl gene was amplified by the polymerase chain reaction using the following two synthetic oligonucleotides as primers: 5′-CGCGCGTCGACATAATTATTATTTAGTGATGATGATGATGATGGATATAGTCAAAGCG-3′ and 5′-GCGGAGATCTAACTCACTG-3′. The first oligonucleotide hybridizes to the 5′ part of the tpl gene and contains six histidine codons (italics), in frame, just before the stop codon and a new SalI site (underlined). The second oligonucleotide hybridizes to the unique BglII site (underlined) in the tpl gene upstream to the transcriptional start point. The resulting 1.9-kilobase pair polymerase chain reaction product was cut with BglII andSalI and subcloned into the BamHI-SalI sites of the expression vector pTZ18U (Bio-Rad) to generate pTZTPL-His. The mutants were prepared by polymerase chain reaction from pTZTPL-His using the QuikChangeTM Site-directed Mutagenesis Kit from Stratagene and the following primer pairs: R100Ta, 5′-CCTACTCACCAGGGGACCGGCGCAGAAAACCTG-3′; R100Tb, 5′-CAGGTTTTCTGCGCCGGTCCCCTGGTGAGTAGG-3′; V283Ra, 5′-CTTCGTACACACGGACTAAC-3′; and V283Rb, 5′-GTTAGTCCGTGTGTACGAAG-3′. The insertion of the histidine codons and the mutations was verified by cycle sequencing (Sequi Therm Long-Read Cycle Sequencing Kit-LC, Epicentre Technologies) with fluorescent primers using a DNA sequencer (LI-COR). E. coli SVS370 cells were used as host for the pTZTPL-His and the mutant plasmids. The cells, grown as described previously (22Chen H. Gollnick P. Phillips R.S. Eur. J. Biochem. 1995; 229: 540-549Crossref PubMed Scopus (46) Google Scholar), were thawed and suspended in 5 ml of Buffer A (50 mm sodium phosphate, 300 mm NaCl, 10 mm imidazole, 1 mmphenylmethylsulfonyl fluoride, 5 mm 2-mercaptoethanol, 0.1 mm PLP, pH 8.0) per gram of wet weight. The cells were disrupted by three passages through a French press. Cell debris was removed by centrifugation at 25,000 × g at 4 °C for 30 min. The supernatant was passed through a 0.22-μm filter and directly applied onto a 13 × 1-cm column containing 2–3 ml of nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with Buffer A. The column was washed with Buffer A containing 20 mmimidazole until A 280 of the flow-through solution was below 0.01. The TPL protein was then eluted with a 30-ml gradient from 20 to 250 mm imidazole in Buffer A. The pooled TPL fractions were dialyzed extensively against 0.1m potassium phosphate, pH 7.0, containing 0.1 mm PLP, 1 mm EDTA, and 5 mm2-mercaptoethanol. Purified wild-type and mutant TPLs were stable at least for 1 year when stored at −70 °C in the same buffer at a concentration of 2–5 mg/ml. All preparations were pure as indicated by SDS-polyacrylamide gel electrophoresis (10–15% PHAST-gel from Amersham Pharmacia Biotech). The concentration of purified TPLs was determined photometrically ( E2781% = 8.37; Ref. 5Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar) assuming a subunit molecular mass of 52.3 kDa (13Antson 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 (130) Google Scholar) which takes into account the molecular mass of the His6 tag (0.84 kDa). The PLP content of the enzymes was determined from the absorption spectrum of the enzyme in 0.1 m NaOH, assuming ε388 = 6600m−1 cm−1 (23Peterson E.A. Sober H.A. J. Am. Chem. Soc. 1954; 76: 169-175Crossref Scopus (306) Google Scholar). Prior to recording absorption spectra, the stock enzyme was incubated with 0.5 mm PLP for 1 h at 30 °C and then separated from excess PLP on a short desalting column (NAPTM 5, Amersham Pharmacia Biotech) equilibrated with 50 mm potassium phosphate, pH 8.0. Absorption spectra were measured with a 8453 UV-visible diode-array spectrophotometer from Hewlett-Packard. The activity of the TPLs toward various amino acid substrates was measured using the coupled assay with lactate dehydrogenase and NADH previously described for tryptophan indole-lyase (24Morino Y. Snell E.E. Methods Enzymol. 1970; 17A: 439-446Crossref Scopus (68) Google Scholar). The standard assay mixture contained 50 mm potassium phosphate, pH 8.0, 5 mm2-mercaptoethanol, 50 μm PLP, 0.2 mm NADH, 24 units of lactate dehydrogenase from bovine heart (Sigma), and varying concentrations of amino acid substrate in a final volume of 1 ml at 25 °C. The reaction was initiated by the addition of TPL and followed by the decrease in absorbance at 340 nm. Steady-state kinetic values of k cat and K m were obtained by fitting the data to the Michaelis-Menten equation using ORIGIN software (Microcal Software). Mutant TPLs and wild-type enzyme were incubated in 50 mm potassium phosphate, pH 8.0, with different amino acids as substrates. Samples were withdrawn at different times and immediately deproteinized with 1m perchloric acid (25Kochhar S. Christen P. Eur. J. Biochem. 1992; 203: 563-569Crossref PubMed Scopus (35) Google Scholar). After derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (26Marfey P. Carlsberg Res. Commun. 1984; 49: 591-596Crossref Scopus (1373) Google Scholar), the reaction samples were loaded onto a reverse-phase high pressure liquid chromatography column (Aquapore RP-300; 250 × 4.6-mm). Both pyridoxamine 5′-phosphate (PMP), produced by the single turnover half-reaction of transamination, and the products of racemization can be separated and detected photometrically at 340 nm by this sensitive assay (27Kochhar S. Christen P. Eur. J. Biochem. 1988; 175: 433-438Crossref PubMed Scopus (26) Google Scholar). Alternatively, the increase in absorbance at 325 nm was used to follow the production of PMP. Protein was eliminated from the reaction mixture prior to chromatography by precipitation with 1m perchloric acid (25Kochhar S. Christen P. Eur. J. Biochem. 1992; 203: 563-569Crossref PubMed Scopus (35) Google Scholar). Thin layer chromatography was performed on pre-coated silica gel plates SIL G-25 from Macherey-Nagel in n-pentyl formate/chloroform/formic acid (20:70:10, v/v). A slightly alkaline solution of bromcresol green (0.02% in ethanol) was used to develop the chromatogram. The acids appeared as yellow spots on a blue background (28Hansen S.A. J. Chromatogr. 1976; 124: 123-126Crossref PubMed Scopus (13) Google Scholar). Formic acid was determined with the formate dehydrogenase assay. A kit from Boehringer Mannheim was used according to the supplier's protocol. Briefly, mutant TPLs were incubated at 25 °C in 50 mm potassium phosphate, pH 8.0, with l-aspartate as substrate. Samples were withdrawn at different times and immediately deproteinized with perchloric acid. Formate was quantitated by the increase in absorbance at 340 nm due to NAD+ reduction. The crystal structure of the holoenzyme complex with the substrate analog 3-(4-hydroxyphenyl) propionic acid (Brookhaven Protein Data Bank, code2TPL) was used as parent structure. The substrate analog was replaced by l-tyrosine, and the external aldimine form 1(Scheme FS1) was created by introducing a double bond between C4′ and the nitrogen atom of the substrate. Removal of the Cα-hydrogen, change of the hybridization of the Cα atom fromsp 3 to sp 2, and subsequent minimization led to the quinonoid intermediate 2. Molecular dynamics simulations of this intermediate were performed using the Discover program (Molecular Simulations) with the consistent valence force field. The cell multipole method was used instead of a cut-off for the nonbonded interactions. The temperature was set to 400 K. All hydrogen atoms and explicit water molecules were included in the simulations with time steps of 1 fs. At the beginning, the whole system was minimized for 2000 steps. The outer shell was then kept fixed, and another 2000 steps of minimization were applied. This was followed by a molecular dynamics simulation, which was initialized at 400 K for 1000 fs. After this initialization, the outer shell was again kept fixed. The simulation was continued for a total time of 20 ps. Every 100 fs the potential energy was analyzed. Within each picosecond, only the structure with the lowest potential energy was stored, resulting in a total of 20 low energy structures. All these 20 structures were then minimized for 2500 steps. The resulting minimized structures were found to be generally quite similar, and one of these corresponding to the average structure was chosen as starting point for all further simulations. The modeled structure of the wild-type enzyme withl-aspartate as substrate was obtained by replacement ofl-tyrosine and applying the same minimization-dynamics procedure as for the wild-type structure with l-tyrosine as substrate. To model the double mutant enzyme, we replaced Arg-100 by a threonine and Val-283 by an arginine residue and applied again the minimization-dynamics procedure. In order to change the substrate specificity of TPL in favor of dicarboxylic amino acids, we compared TPL with AspAT using homology modeling and molecular dynamic simulations. The specificity of AspAT for dicarboxylic amino acids and oxo acids seems to be based primarily on the salt bridge-hydrogen bond interaction of the side chain of Arg-292 (of the adjacent subunit) with the distal carboxylate group of these substrates (12Kirsch 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 (420) Google Scholar). In agreement with this notion, Arg-292 is conserved among all AspATs (29Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1989; 186: 249-253Crossref PubMed Scopus (124) Google Scholar). Since the sequence identity between AspAT and TPL is too low (23%) to allow the use of standard alignment algorithms, comparison of their three-dimensional structures (13Antson 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 (130) Google Scholar, 30Jäger J. Moser M. Sauder U. Jansonius J.N. J. Mol. Biol. 1994; 239: 285-305Crossref PubMed Scopus (174) Google Scholar) by superposition (Fig.1) and with the program DALI (Fig.2) was used to identify in TPL the residue corresponding to Arg-292 in AspAT. Val-283 in TPL seems to occupy the same position as Arg-292 in AspAT. Another significant difference in the active sites of these two enzymes is the replacement of a residue interacting with the phosphate group of the coenzyme. Arg-100 in TPL apparently corresponds to Thr-109 in AspAT which is also conserved among all AspATs (Figs. 1 and 2; Ref. 29Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1989; 186: 249-253Crossref PubMed Scopus (124) Google Scholar).Figure 2Sequence alignment of C. freundiiTPL and E. coliAspAT with the program DALI (Ref.31Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3545) Google Scholar,32Holm L. Sander C. Nucleic Acids Res. 1997; 25: 231-234Crossref PubMed Scopus (357) Google Scholar; accessible through the World Wide Web at the following on-line address: http://www2.ebi.ac.uk/dali/ ). Identical residues are indicated in bold. Residues that belong to α-helices, β-strands, and β-turns are marked by the letters h, s, and t, respectively. The amino acid residues in TPL corresponding to Thr-109 and Arg-292 in AspAT are marked with anasterisk.View Large Image Figure ViewerDownload (PPT) Molecular modeling showed that the quinonoid adduct ofl-aspartate and PLP can be sterically accommodated in the active site of wild-type TPL (Fig. 3). However, the orientation of the leaving group of the substrate relative to the planar coenzyme-substrate adduct did not appear to be optimum for a β-elimination reaction. Positively charged Arg-100 in the hydrophobic active site of TPL interacted with the distal carboxylate group of dicarboxylic substrates and thus perturbed the required orthogonal orientation of the plane defined by Cα, Cβ, and Cγ of the amino acid substrate relative to the plane defined by the π system of the coenzyme-substrate adduct including Cβ (Scheme FS1; Ref.33Dunathan H.C. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 712-716Crossref PubMed Scopus (339) Google Scholar). This notion agrees with previous studies by Faleev et al. (34Faleev N.G. Ruvinov S.B. Demidkina T.V. Myagkikh I.V. Gololobov M.Y. Bakhmutov V.I. Belikov V.M. Eur. J. Biochem. 1988; 177: 395-401Crossref PubMed Scopus (38) Google Scholar) who have reported that aspartic and glutamic acid are not substrates but, in view of the low hydrophobicity of their side chains, anomalously strong inhibitors of TPL (K i = 3.5 and 5.0 mm, respectively). We concluded that the introduction of an arginine residue into position 283 of TPL together with the substitution of Arg-100 with an uncharged residue, i.e. the double mutation R100T/V283R, might mimic the binding site for dicarboxylic substrates of AspAT and thus result in a corresponding alteration in the substrate specificity of TPL. The C-terminal His6 tag did not interfere with the β-elimination activity of the enzyme (Table I). The His-tagged TPL R100T/V283R enzyme and the single mutant TPL R100T were purified and used for analysis. The single mutant V283R enzyme, however, could not be expressed as soluble protein. The PLP content of the mutant proteins was found to be 1 mol/mol of subunit, as has been shown previously for wild-type TPL (36Kazakov V.K. Myagkikh I.V. Tomina I.I. Demidkina T.V. Biokhimiya. 1987; 52: 1319-1323Google Scholar). The UV-visible spectrum of the PLP form of the mutant enzymes is almost identical to that of the wild-type enzyme. Apparently, the topochemistry of the PLP-binding site is not significantly altered by the mutations.Table IKinetic parameters for β-elimination reaction of tyrosine phenol-lyase wild-type, R100T, and R100T/V283R mutantsSubstrateaThe concentration ranges of the tested amino acids were as follows: 10–200 mm for dicarboxylic amino acids; 0.2–2 mm for l-tyrosine.Wild-type enzymeR100TR100T/V283Rk catK mk cat/K mk catK mk cat/K mk catK mk cat/K ms−1mmm−1s−1s−1mmm−1s−1s−1mmm−1s−1l-Tyrosine3.70.21.85 × 1040.54bOwing to the low solubility of tyrosine (up to 2 mm), these values are less certain than those of the TPL R100T/V283R.8.3bOwing to the low solubility of tyrosine (up to 2 mm), these values are less certain than those of the TPL R100T/V283R.65.1bOwing to the low solubility of tyrosine (up to 2 mm), these values are less certain than those of the TPL R100T/V283R.0.110.32343.8l-AspartateActivity below detection0.035560.630.21543.9l-GlutamateActivity below detection0.08155.40.105.318.9l-2-AminoadipateActivity below detection0.095cSaturation was not apparent within the concentration range tested. Values measured at a substrate concentration of 120 mm.—cSaturation was not apparent within the concentration range tested. Values measured at a substrate concentration of 120 mm.—0.19cSaturation was not apparent within the concentration range tested. Values measured at a substrate concentration of 120 mm.—cSaturation was not apparent within the concentration range tested. Values measured at a substrate concentration of 120 mm.—3-Chloro-l-alanine3.0dFrom Ref. 43.1.7dFrom Ref. 43.1.8 × 1031.1319591.134625Values were determined in 50 mm potassium phosphate, pH 8.0, at 25 °C. Pyruvate production followed by the coupled assay with lactate dehydrogenase and NADH. In control reactions with 50 μm PLP but without enzyme, no activity was detected in the case of any substrate. All enzymes carry a C-terminal His6tag. The activity of the tagged wild-type enzyme towardl-tyrosine corresponds closely to that of the untagged wild-type enzyme (35Chen H. Demidkina T.V. Phillips R.S. Biochemistry. 1995; 34: 12276-12283Crossref PubMed Scopus (61) Google Scholar).a The concentration ranges of the tested amino acids were as follows: 10–200 mm for dicarboxylic amino acids; 0.2–2 mm for l-tyrosine.b Owing to the low solubility of tyrosine (up to 2 mm), these values are less certain than those of the TPL R100T/V283R.c Saturation was not apparent within the concentration range tested. Values measured at a substrate concentration of 120 mm.d From Ref. 43Sundararaju B. Antson A.A. Phillips R.S. Demidkina T.V. Barbolina M.V. Gollnick P. Dobson G.G. Wilson K.S. Biochemistry. 1997; 36: 6502-6510Crossref PubMed Scopus (74) Google Scholar. Open table in a new tab Values were determined in 50 mm potassium phosphate, pH 8.0, at 25 °C. Pyruvate production followed by the coupled assay with lactate dehydrogenase and NADH. In control reactions with 50 μm PLP but without enzyme, no activity was detected in the case of any substrate. All enzymes carry a C-terminal His6tag. The activity of the tagged wild-type enzyme towardl-tyrosine corresponds closely to that of the untagged wild-type enzyme (35Chen H. Demidkina T.V. Phillips R.S. Biochemistry. 1995; 34: 12276-12283Crossref PubMed Scopus (61) Google Scholar). Wild-type TPL in the presence of l-tyrosine exhibits a visible absorbance peak at about 500 nm attributable to the quinonoid coenzyme-substrate adduct 2 (Scheme FS1; Ref. 8Kumagai H. Kashima N. Yamada H. Biochem. Biophys. Res. Commun. 1970; 39: 796-801Crossref PubMed Scopus (56) Google Scholar). Some other amino acids such as l- and d-alanine,l-phenylalanine, l-aspartic acid,l-methionine, and l-homoserine, which are not substrates for β-elimination, form stable quinonoid intermediates with wild-type TPL (34Faleev N.G. Ruvinov S.B. Demidkina T.V. Myagkikh I.V. Gololobov M.Y. Bakhmutov V.I. Belikov V.M. Eur. J. Biochem. 1988; 177: 395-401Crossref PubMed Scopus (38) Google Scholar). TPL R100T also produced stable quinonoid intermediates upon addition of these amino acids. However, with TPL R100T/V283R no detectable quinonoid adduct was observed in the presence of any amino acid including l-tyrosine. TPL R100T/V283R and TPL R100T were tested for β-elimination activity towardl-tyrosine and dicarboxylic amino acids of various lengths (Table I). The k cat value of TPL R100T/V283R toward l-tyrosine was decreased 30-fold as compared with wild-type TPL without significant change in the K mvalue. When TPL R100T/V283R was tested for activity toward dicarboxylic amino acids using the coupled assay with lactate dehydrogenase and NADH, pyruvate was detected in the reaction mixtures. Thin layer chromatographic analyses confirmed the production of pyruvate. A yellow spot, the R f value of which was the same as that of authentic pyruvate, was detected on the plate as the unique and invariable oxo acid product of the enzymic reactions with all dicarboxylic substrates. No 2-oxobutyric acid, which possibly might have been produced by a γ-elimination reaction ofl-glutamate, was detected. The expected products of the β-elimination reaction of the dicarboxylic substrates l-aspartate,l-glutamate, and l-2-aminoadipate are pyruvate, ammonia, and the monocarboxylic acids formate, acetate, and propionate, respectively. In the case of l-aspartate, formate was identified and determined using the coupled assay with formate dehydrogenase and NAD+. Equimolar amounts of pyruvate and formate were detected (Table II). Thus, TPL R100T/V283R catalyzes, in contrast to the wild-type enzyme, the β-elimination reaction of dicarboxylic substrates at least as efficiently or, in the case of l-aspartate, even two times faster than that of l-tyrosine (k cat= 0.21 s−1; Table I). The K m value of TPL R100T/V283R for the β-elimination reaction withl-glutamate was significantly lower than theK m values with l-aspartate andl-2-aminoadipate. It seems that l-glutamate has the optimum size for binding among these dicarboxylic substrates. TPL R100T also catalyzed the β-elimination reaction of the dicarboxylic amino acids l-aspartate, l-glutamate, andl-2-aminoadipate; however, the reaction was up to six times slower than that with the double mutant enzyme. TheK m values were also higher (up to 3 times, in the case of l-glutamate) as compared with the double mutant enzyme. Furthermore, the TPL R100T-catalyzed β-elimination reaction of l-tyrosine was five times faster than the TPL R100T/V283R-catalyzed reaction.Table IIStoichiometry of the β-elimination reaction of tyrosine phenol-lyase R100T/V283R with l-aspartateTime of reactionProductsPyruvateaPyruvate was measured using the coupled a" @default.
• W2061178897 created "2016-06-24" @default.
• W2061178897 creator A5076302808 @default.
• W2061178897 creator A5076865729 @default.
• W2061178897 creator A5087072437 @default.
• W2061178897 creator A5087920247 @default.
• W2061178897 date "1999-01-01" @default.
• W2061178897 modified "2023-09-28" @default.
• W2061178897 title "Conversion of Tyrosine Phenol-lyase to Dicarboxylic Amino Acid β-Lyase, an Enzyme Not Found in Nature" @default.
• W2061178897 cites W103646132 @default.
• W2061178897 cites W141150985 @default.
• W2061178897 cites W1414256803 @default.
• W2061178897 cites W1506914419 @default.
• W2061178897 cites W1597078415 @default.
• W2061178897 cites W1667751859 @default.
• W2061178897 cites W1773196754 @default.
• W2061178897 cites W1861199552 @default.
• W2061178897 cites W1967229255 @default.
• W2061178897 cites W1967414045 @default.
• W2061178897 cites W1967999247 @default.
• W2061178897 cites W1968592972 @default.
• W2061178897 cites W1974582518 @default.
• W2061178897 cites W1980585710 @default.
• W2061178897 cites W1984210216 @default.
• W2061178897 cites W1995237081 @default.
• W2061178897 cites W2003039115 @default.
• W2061178897 cites W2003300391 @default.
• W2061178897 cites W2003764452 @default.
• W2061178897 cites W2004681183 @default.
• W2061178897 cites W2010167159 @default.
• W2061178897 cites W2021388552 @default.
• W2061178897 cites W2022058405 @default.
• W2061178897 cites W2044672033 @default.
• W2061178897 cites W2046378950 @default.
• W2061178897 cites W2049553542 @default.
• W2061178897 cites W2051291172 @default.
• W2061178897 cites W2056200760 @default.
• W2061178897 cites W2057795357 @default.
• W2061178897 cites W2060177102 @default.
• W2061178897 cites W2062880866 @default.
• W2061178897 cites W2075878659 @default.
• W2061178897 cites W2080894377 @default.
• W2061178897 cites W2083446995 @default.
• W2061178897 cites W2091471707 @default.
• W2061178897 cites W2092163817 @default.
• W2061178897 cites W2092859952 @default.
• W2061178897 cites W2121524599 @default.
• W2061178897 cites W2137345862 @default.
• W2061178897 cites W2139632794 @default.
• W2061178897 cites W2156613138 @default.
• W2061178897 cites W2168943734 @default.
• W2061178897 cites W2326842020 @default.
• W2061178897 cites W2328430051 @default.
• W2061178897 cites W4235403728 @default.
• W2061178897 cites W88129378 @default.
• W2061178897 doi "https://doi.org/10.1074/jbc.274.3.1320" @default.
• W2061178897 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9880502" @default.
• W2061178897 hasPublicationYear "1999" @default.
• W2061178897 type Work @default.
• W2061178897 sameAs 2061178897 @default.
• W2061178897 citedByCount "25" @default.
• W2061178897 countsByYear W20611788972012 @default.
• W2061178897 countsByYear W20611788972013 @default.
• W2061178897 countsByYear W20611788972016 @default.
• W2061178897 countsByYear W20611788972017 @default.
• W2061178897 countsByYear W20611788972018 @default.
• W2061178897 countsByYear W20611788972019 @default.
• W2061178897 crossrefType "journal-article" @default.
• W2061178897 hasAuthorship W2061178897A5076302808 @default.
• W2061178897 hasAuthorship W2061178897A5076865729 @default.
• W2061178897 hasAuthorship W2061178897A5087072437 @default.
• W2061178897 hasAuthorship W2061178897A5087920247 @default.
• W2061178897 hasBestOaLocation W20611788971 @default.
• W2061178897 hasConcept C181199279 @default.
• W2061178897 hasConcept C185592680 @default.
• W2061178897 hasConcept C2776165026 @default.
• W2061178897 hasConcept C2777269803 @default.
• W2061178897 hasConcept C515207424 @default.
• W2061178897 hasConcept C55493867 @default.
• W2061178897 hasConceptScore W2061178897C181199279 @default.
• W2061178897 hasConceptScore W2061178897C185592680 @default.
• W2061178897 hasConceptScore W2061178897C2776165026 @default.
• W2061178897 hasConceptScore W2061178897C2777269803 @default.
• W2061178897 hasConceptScore W2061178897C515207424 @default.
• W2061178897 hasConceptScore W2061178897C55493867 @default.
• W2061178897 hasIssue "3" @default.
• W2061178897 hasLocation W20611788971 @default.
• W2061178897 hasLocation W20611788972 @default.
• W2061178897 hasOpenAccess W2061178897 @default.
• W2061178897 hasPrimaryLocation W20611788971 @default.
• W2061178897 hasRelatedWork W1496234280 @default.
• W2061178897 hasRelatedWork W1644178486 @default.
• W2061178897 hasRelatedWork W1942554446 @default.
• W2061178897 hasRelatedWork W1999543479 @default.
• W2061178897 hasRelatedWork W2004312381 @default.
• W2061178897 hasRelatedWork W2087917372 @default.
• W2061178897 hasRelatedWork W2168498879 @default.
• W2061178897 hasRelatedWork W2529482336 @default.

• next