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The Interaction of Escherichia Coli Tryptophanase with Various Amino

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The Interaction of Escherichia Coli Tryptophanase with Various Amino J. Biochem. 82, 733-745 (1977) The Interaction of Escherichia coli Tryptophanase With Various Amino Acids and Their Analogs Active Site Mapping1 Takehiko WATANABE2 and Esmond E. SNELL2 Department of Biochemistry, University of California, Berkeley, California 94720, U.S.A. Received for publication, April 9, 1977 Tryptophanase [TPase, L-tryptophan indole-lyase, EC 4.1.99.1] from Escherichia coli B was studied extensively with respect to its interaction with various amino acids and their analogs. Several new substrates were found; these include ƒÀ-chloro-L-alanine, L-cysteine sulfinate, S-benzyl-L-cysteine, O-benzyl-L-serine, L-threonine, L-allothreonine, and the two ƒÀ-methyl-L- tryptophans. Formation of S-hydroxyethyl-L-cysteine from L-serine and 2-mercaptoethanol was also demonstrated. Most amino acids tested, including these substrates, were competitive inhibitors of the TPase reaction with L-tryptophan as the substrate. The Kl values differed so markedly among various amino acids that it was possible to draw conclusions concerning the topography of the active site of TPase. The indole ring or other electronegative group on the ƒÀ-carbon atom, together with a carboxyl group on the a-carbon atom , is necessary for oriented binding to the active site of TPase; a free a-amino group is also required for substrate activity, but does not contribute greatly to the binding interactions. Most amino acids and their analogs induced an absorption peak near 500 run, previously assigned to quinoidal structure formed by labilization of the a-proton of the enzyme-bound Schiff's base between PLP and amino acid. Amino acids that have an asymmetric carbon at the ƒÀ-position (L-threonine, L-allothreonine, L-isoleucine, ,ƒÀ-methyl-L-tryptophan) and some other amino acids (L-valine, phenylglycine, glycine) did not evoke this 500 nm peak. Bromopyruvate proved to be a very potent inactivator of TPase. Pyridoxal 5'-phosphate completely prevented this inactivation, but inhibitor amino acids did not. One SH group per subunit of TPase was blocked by treatment with bromopyruvate. The cysteine residue that reacts is probably located in the peptide T-18-1 isolated by Kagamiyama et a!. [J. Biol. Chem. 247, 1571-1575 (1972)]. TPCK inactivated TPase in a manner similar to bromopyruvate. 1 Dedicated to Dr. Katashi Ichihara on his 80th birthday. This study was supported in part by grants AM 1448 and Al 1575 from the National Institutes of Health, U.S. Public Health Service. 2 Present address: Department of Pharmacology , Osaka University School of Medicine, Kita-ku, Osaka 530. 3 Present address: Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712, U.S.A. Abbreviations: TPase, tryptophanase; PLP, pyridoxal 5'-phosphate; DTNB, 5, 5'-dithiobis(2-nitrobenzoic acid); TPCK, N-tosyl-L-phenylalanylchloromethane; TLCK, N-tosyl-L-lysylchloromethane; Bicine, N, N-bis(2-hydroxy ethyl)glycine; LDH, lactic dehydrogenase. Vol. 82, No. 3, 1977 733 734 T. WATANABE and E.E. SNELL Tryptophanase from E. coli B/lt7A is one of the This paper describes extensive studies of amino most extensively studied PLP-dependent enzymes acids and their analogs as possible substrates or (1). It catalyzes a large number of reactions as inhibitors of TPase and their effects on absorption follows. spectra of the enzyme. It also describes the in- activation of TPase by bromopyruvate and TPCK. RCH2CHNH2COOH+H2O•¨. RH+CH3C0COOH+NH3 (1 ) MATERIALS AND METHODS where R=indolyl or substituted indolyl groups, Enzyme Preparation-Apotryptophanase was NH2, OR' or SR' (R'=H, CH3, CH2CH3), etc., purified from E. coli B/lt7A according to the (i.e., a, /l-elimination reactions) (2, 3). procedure reported in the previous paper (4). The apoenzyme could be stored in crystalline form RCH2CHNH2COOH+R'H -> in 60% ammonium sulfate solution for at least R'CH2CHNH2COOH+RH (2) 3 months without detectable loss of activity. Holoenzyme was prepared as described by Morino where R'H=indole or a substituted indole and R is as designated in Eq. 1 (i.e., ƒÀ-replacement and Snell (6), except that 0.1 m i dithiothreitol was used in place of 2 mm 2-mercaptoethanol. reactions) (2, 3). Enzyme Assays-The enzyme was routinely Indole+CH3COCOOH+NH3•¨ assayed by measuring indole formed from L- L-Tryptophan+H20 (3) tryptophan using a slight modification of the published procedure (9). Glutathione (120 leg per where indole can be replaced by substituted indoles a tube) was replaced by 0.1 mm dithiothreitol and (i.e., L-tryptophan synthesis via reversal of the the concentration of PLP was increased to 0.1 mm. TPase reaction) (4, 5). In studies of inhibition kinetics, reaction mixtures containing inhibitors were preincubated for 10 min RCH2CHNHZCOOHD2O? at 37•Ž; the reaction was then initiated by the H2O H20 addition of holoenzyme. Pyruvate and ƒ¿-keto RCH2CDNH2COOH (4) butyrate was assayed according to Friedman and Haugen (10), or occasionally by the LDH method (i.e., labilization of the ƒ¿-hydrogen of a variety of (11). Ammonia produced from ƒÀ-chloro-L-alanine amino acids, called quasi-substrates (6), that do was analyzed by Nessler's method (12). Sulfur not undergo reactions 1-3). dioxide in a reaction with L-cysteine sulfinate as a Several amino acids have been reported to substrate was determined by the procedure of West interact with TPase on the basis of inhibition and Gaeke (13). ƒÀ-Methyl-L-tryptophan was kinetics or their effects on the absorption spectra assayed by the method of Udenfriend et al. (14). (6). All were competitive inhibitors of TPase The formation of S-hydroxyethyl-L-cysteine. from reactions, while some were also substrates; most of L-serine and 2-mercaptoethanol was followed with them induced the formation of a characteristic an amino acid analyzer as described by Miles et a!. absorption band near 500 nm. More thorough (15); its identity was confirmed by paper chro studies of additional substrate and/or inhibitor matography using pyridine: water=8 : 2 (v/v). amino acids would assist in understanding the Absorption Spectroscopy-Absorption spectra nature of the substrate binding site, in the inter were taken with a Cary 14 spectrophotometer at pretation of absorption spectra, and in the design room temperature. Mixtures contained 100 ƒÊmol of affinity labels for TPase. Studies of the active of potassium phosphate buffer, pH 7.8, 2 ƒÊemol of site of this interesting enzyme are rather limited. EDTA, 0.1 ƒÊmol of dithiothreitol, 1-2 mg of holo The role of SH groups was first studied by Wada TPase, and appropriate amounts of amino acids in et al. (7); Morino and Snell (8) later demonstrated a total volume of 1.0 ml. that titration of two SH-groups per subunit Inactivation of Tease by Bromopyruvate and (55,000 daltons) of TPase with DTNB was accom TPCK-Prior to inactivation studies, TPase was panied by complete loss of enzyme activity. passed through a small column of Sephadex G-25 J. Biochem. ACTIVE[SITE MAPPING OF Escherichia coli TRYPTOPHANASE 735 (0.9 x 15 cm) equilibrated with 0.1 M potassium prepared by exposure of a chromatogram to Kodak phosphate buffer, pH 7.5, and 2 mm EDTA to X-ray film for appropriate periods. The peptides remove dithiothreitol. Appropriate amounts of were further purified by high-voltage paper electro bromopyruvate were added to TPase in 0.1 M phoresis at pH 1.7 (formic acid: acetic acid: water= Bicine buffer, pH 8.0, 0.1 M KC1, and 2 mm EDTA, 5 : 15 : 80 (v/v)) at 2,000 volts for 2 h and paper and at the indicated times aliquots were diluted chromatography using 1-butanol: acetic acid: into assay mixtures to measure the remaining water=200 : 30 : 75 (v/v) as a solvent. Digestion activities. Since the assay mixture contained 0.1 with carboxypeptidase A was conducted by the mm dithiothreitol, unreacted bromopyruvate was method of Kagamiyama et al. (19). destroyed instantaneously. The inactivation by Chemicals-ƒÀ-Methyltryptophans were a kind TPCK was conducted similarly, except that 10 gift from Dr. H. Floss, Purdue University. Sodium methanol was included in the incubation mixture to pyruvate-2-14C was obtained from Amersham/ ensure solubility of the TPCK. Methanol in Searle. Bromopyruvate-2-14C was synthesized by amounts up to 15 % on the assay mixture had no direct bromination of sodium pyruvate-2-14C effect on TPase activity. diluted with cold redistilled pyruvic acid by the Titration of SH Groups with DTNB-The num method of Meloche (20). The specific radioactivity ber of SH groups on native and bromopyruvate of the product was determined from the amount or TPCK-treated TPase was determined by the of hydroxypyruvate produced by alkaline treatment DTNB procedure as used by Morino and Snell (8). of the bromopyruvate (20). Other chemicals were Identification of the Residue Modified by of analytical grade and were used without further Bromopyruvate-TPase treated with bromopyru purification. vate-2-14C was reduced with 20mM NaBH4 according to the method of Meloche (16) to convert the car RESULTS boxyketoethyl moiety introduced into the protein to the stable carboxyhydroxyethyl moiety. The Additional Substrates of Tryptophanase reduced enzyme was hydrolyzed in 6 N HCl at It was reported earlier that amino acids with 110•Ž for 20 h and the hydrolysate was analyzed electronegative groups of appropriate structure at on a Beckman 120 C amino acid analyzer. A new their ƒÀ-position were substrates of TPase (2, 3, 6). Several additional amino acids that act as substrates peak was eluted from the long column (50 cm) at are listed in Table I. All of these reactions pro 56.5•Ž by pH 3.22 buffer at 41 min; aspartic acid was eluted at 45.5 min under the same conditions.
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