Stereochemistry and Mechanism of Reactions Catalyzed by Tryptophan Synthetase and Its P2 Subunit*
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Stereochemistry and Mechanism of Reactions Catalyzed by Tryptophan Synthetase and Its p2 Subunit* (Received for publication, December 27, 1977, and in revised form, February 13, 1978) Ming-Daw Tsai, Erwin Schleicher, Rowe11 Potts, George E. Skye, and Heinz G. Floss From the Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 The synthesis of tryptophan from serine and indole ,&Mercaptoethanol + L-serine + S-hydroxyethyl-L-cysteine (4) or indoleglycerol phosphate catalyzed by native tryp- /3-Mercaptoethanol + L-serine + pyridoxal phosphate tophan synthetase or PZ protein is shown to proceed (5) stereospecifically with retention of configuration at Cg. + S-pyruvyl mercaptoethanol + pyridoxamine phosphate In the a$ elimination reaction of serine to give pyru- As in the case of other pyridoxal phosphate-containing vate and ammonia catalyzed by the fiz protein, the enzymes which carry out p replacement and/or a$ elimina- hydrogen from C, is transferred intramolecularly and tion reactions of amino acids, the reaction sequence leads via without exchange with solvent protons to Cg, where it a series of aldimine and ketimine complexes between the Downloaded from replaces the OH group with net retention of configu- substrate amino acid and the cofactor to an enzyme-bound ration. In a competing reaction, an abortive transami- Schiff s base between pyridoxal phosphate and cr-aminoacrylic nation in the presence of mercaptoethanol to give pyr- acid as a universal intermediate. This intermediate can follow idoxamine and S-pyruvyl mercaptoethanol, the same proton is transferred to the extent of 70% to C-4’ of the various reaction paths, giving the different observed products. cofactor when the reaction is carried out in DzO. To- In this paper we report results which clarify various ster- www.jbc.org gether with the finding of Dunathan and Voet (Duna- eochemical aspects of these reactions, define the geometry of than, H. G. and Voet, J. G. (1974) Proc. Natl. Acad. Sci. the coenzyme.substrate complex in the active site and throw U. S. A. 71, 3888) that the cofactor is protonated from some light on several of the protonation steps involved in the si face, these data fully define the geometry of the these reactions. Some of the results have been communicated substrate l coenzyme complex and the position of an in preliminary form (2, 3). at unknown institution, on September 29, 2009 essential base relative to it. Since no isotope effect was EXPERIMENTAL PROCEDURES observed in the protonation of C, of tryptophan syn- Materials-The chemicals used were of reagent grade or of the thesized from indole and serine in 50% DzO, the base highest purity commercially available and they were used without must be monoprotic. The known inability of the enzyme further purification. All enzymes obtained commercially were pur- to degrade tryptophan by a$ elimination is not due to chased from Sigma. L-[ U-‘“C]Serine (159 mCi/mmol), L-[3-“‘Cltryp- inability to remove H, of tryptophan; native enzyme tophan (-45 mCi/mmol), and [2-‘“Cl acetate (-50 mCi/mmol) were and j3~ protein catalyze a hydrogen exchange of tryp- purchased from Amersham/Searle. Reference indolmycin was a gift tophan at a rate of20% and 14%, respectively, of that of from Charles Pfizer and Co. (2S,3R)- and (2S.35’)~[3-“Hjserine were prepared as described previously (4) whereas (2S,3R)- and (2S,3S)- tryptophan synthesis. [3-‘H$“H]serine were prepared analogously using D20 as the solvent in the fmst step. DL-[2-‘H]Serine and DL-(3R)-[2-‘H, 3-“Hlserine were synthesized from DL-serine and (2S,3R)-[3-“Hlserine, respectively, according to the procedures of Miles and McPhie (5). Double-labeled Tryptophan synthetase (EC 4.1.2.20) (1) is a tetrameric samples were prepared by mixing the appropriate single-labeled spe- enzyme containing two pairs of identical subunits, which cies and constancy of their “H/“‘C ratio was usually ascertained by normally catalyzes the reaction: rechromatography of an aliquot in at least one solvent system. Indole%glycerol phosphate + L-serine Streptomyces griseus ATCC 12648 was obtained from the Ameri- (1) can Type Culture Collection and was maintained on slants of Emer- + L-tryptophan + n-glyceraldehyde 3-phosphate son’s agar at 24’C. Enzymes and Assay Procedures-The native tryptophan synthe- Alternatively, the enzyme can also synthesize tryptophan tase was purified from Neurospora crassa according to the procedure from L-serine and indole according to Equation 2: of Meyer et al. (6) and Yanofsky (7). Tryptophan synthetase 01 protein was partially purified from Escherichia coli Bx (S), omitting the final Indole + L-serine + L-tryptophan (‘3 DEAE-cellulose column step, to give preparations of 4.7 IU/mg of protein. Tryptophan synthetase /?z protein was purified from E. coli Its ,L?z subunit which contains pyridoxal phosphate as the AJF’A, according to the procedure of Adachi and Miles (9) and had prosthetic group is unable to catalyze reaction 1, but carries a specific activity of 14.6 IU/mg of protein. Both E. coli mutants were out reaction 2 in addition to a number of other reactions, e.g. kindly provided by Dr. R. Somerville. Protein determinations were (1): carried out by Lowry’s method (10) using serum albumin as standard. Tryptophan synthetase activity was measured by following indole L-Serine + pyruvate + ammonia (3) disappearance calorimetrically in the reaction between indole and _..- _ __--- serine to form tryptophan (11). * This work was supported by the United States Public Health Chromatography-Paper chromatography was carried out on Service through Research Grant GM 18852 from the Institute for Whatman No. 3 MM paper using the following solvent systems: General Medical Sciences and by a postdoctoral fellowship (to E. S.) System A, propanol-l/concentrated NHdOH/water, 6:3:1 (descend- from the Max-Kade-Foundation, New York. The costs of publication ing, RF values: tryptophan 0.70, serine 0.46); System B, butanol-l/ of this article were defrayed in part by the payment of page charges. 88% formic acid/water, 14:3:3 (descending, tryptophan 0.54, serine This article must therefore be hereby marked “aduertisement” in 0.20); System C, 95% ethanol/concentrated NHgOH/water, 80:4:16 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (descending, serine 0.61, lactic acid 0.87); and System D, butanol-l/ 5344 Stereochemistry of Tryptophan Synthetase 5345 glacial acetic acid/water, 2:l:l (descending, serine 0.38, S-hydroxy- incubations from which fumarase had been omitted were carried out ethyl cysteine 0.51). Amino acids were visualized with ninhydrin spray to give malate. reagent (5% ninhydrin in ethanol) and lactic acid was located by Deamination ofSerine-The incubation mixture contained in 1 ml comparison with [ U-“‘Cllactic acid as reference. of 0.1 M potassium phosphate buffer, pH 7.8: 40 pmol of I,-serine Hadioactivity Determinations-Radioactivity on chromatograms samples (0.5 PCi), 10 mM glutathione, 0.2 mu pyridoxal phosphate, 80 was located using a Packard model 7201 radiochromatogram scanner. pmol of NADH, 5 IU of the ,& protein of tryptophan synthetase from The radioactivity of compounds in solution was determined in a E. coli, and excess lactate dehydrogenase from pig heart. For the Beckman LS 100 or LS 250 liquid scintillation counter using 2,5- experiments in columns 1 to 4 of Table II, the “H-labeled serine (100 diphenyloxazole (PPO) and 1,4-bis[2-(methyl-5.phenyloxazolyl)]ben- pCi/pmol) was diluted with nonlabeled L-serine. For the experiment zene (dimethyl POPOP) in toluene or toluene/ethanol as scintillator of column 5, Table II, the (2S,3R)-[3-,‘H]serine was diluted with L- solution. Counting efficiencies and the spillover of 14C into the tritium serine before labeling at the o[ position with deuterium and the channel were determined by internal standardization with [‘“Cltolu- resulting DL mixture was used directly, whereas in the experiment of ene and [“Hltoluene. column 6 the carrier serine consisted of a 64.fold excess of DL- Conversion of Serine into Tryptophan-The serine samples ster- [2-‘Hlserine. The incubation was carried out at 37°C for 4 h and eospecifically tritiated at the /3 position were mixed with L-[ U-‘“Cl- stopped by boiling for 3 min. The decrease in the absorbance at 340 serine and converted into tryptophan with the native tryptophan nm was followed during the incubation. The proteins were separated synthetase from Neurospora crassn, the mixture of (Y and 82 protein from the small molecules by dialysis. The lactate samples were isolated separately from E. coli, or the & protein of tryptophan isolated by paper chromatography in System C and further purified synthetase from l?. coli. The incubation mixture contained in a by acidification with HCI and extraction with ether, with the yields volume of 0.2 ml: Tris/HCl, pH 7.8, 10 pmol; 0.1 pmol or less of serine; varying from 20% to 50%. The resulting lactate samples were then 0.2 pmol of indole (or indoleglycerol phosphate); and 0.25 IU of oxidized to acetate by heating with 12 ml of oxidation mixture (153 enzyme. In cases where a mixture of LY and pZ subunits was used, a 2- mg of KzCrzOi; 24 ml of concentrated HZOr; made up to 100 ml with fold excess of a-protein was added and parallel reactions were carried water) on a steam bath for 20 min under an argon atmosphere (14). out in the presence of 0.2 M NaCl. For the experiments with the PJ The acetate samples were then isolated by steam distillation and Downloaded from protein of tryptophan synthetase, parallel incubations were carried analyzed for chirality by the method of Cornforth et al.