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Indole-3-Glycerolphosphate + L-Serine L-Tryptophan L

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Indole-3-Glycerolphosphate + L-Serine L-Tryptophan L SERINE DEAMINATION BY THE B PROTEIN OF ESCHERICHIA COLI TRYPTOPHAN SYNTHETASE* BY IRVING P. CRAWFORDt AND JUNETSU ITO DEPARTMENT OF MICROBIOLOGY, WESTERN RESERVE SCHOOL OF MEDICINE, CLEVELAND Communicated by Bruno Zimm, January 13, 1964 The biosynthetic tryptophan synthetase of Escherichia coli is a bipartite enzyme.' Mutational loss or alteration of either of this enzyme's protein components results in tryptophan auxotrophy, which is correlated with an inability of the residual enzyme to perform reaction 1.2 Indole-3-glycerolphosphate + L-serine L-tryptophan + D-glyceraldehyde-3-phosphate (1) Both tryptophan synthetase components when isolated possess some enzymatic activity, however. The smaller component, the A protein, inefficiently catalyzes reaction 2,1 while the B protein can catalyze tryptophan formation from indole and serine (reaction 3) at a rate which varies markedly with the ionic environ- ment.3 Indole-3-glycerolphosphate = indole + D-glyceraldehyde-3-phosphate (2) Indole + L-serine -- L-tryptophan (3) Although reactions 2 and 3 can be summed to give reaction 1, tryptophan biosyn- thesis from indoleglycerolphosphate and serine probably takes place at a single enzyme surface, for the A and B proteins complex strongly with each other, and free indole has not been detected during the performance of reaction 1.' Recently the B protein has been purified.4 As expected, this protein shows the absorption spectrum of a pyridoxal phosphate enzyme. The present com- munication documents a second enzymatic activity possessed by the B protein in isolation-pyridoxal phosphate-dependent L-serine deamination (reaction 4). L-serine -o pyruvate + ammonia (4) Evidence suggests that the same enzyme-bound pyridoxal phosphate molecules are involved in reactions 3 and 4. One effect of the A protein in combining with the B protein is to enhance reaction 3 activity while diminishing reaction 4 activity. Materials and Methods.-Purification of the enzyme: The B protein can be obtained in an ultra- centrifugally and electrophoretically monodisperse form by purification from extracts of a mutant (A2) of E. coli lacking the A protein.4 The growth of such a mutant is arrested in the stationary phase by tryptophan deprivation to derepress enzyme synthesis. The purification method is essen- tially similar to one published previously,3 with the addition of a final step, heating at 810 for 3 min in 0.5 M potassium phosphate buffer, pH 7.5. Pure preparations show specific activities of over 2500, representing a 100-fold purification of the activity present in crude extracts. Crystalline A protein was prepared by the method of Henning, Helinski, Chao, and Yanofsky.5 Enzyme assays: Reactions 2 and 3 were assayed as previously described.6 Reaction 1 is assayed spectrophotometrically by observing glyceraldehydephosphate appearance with a coupled glycerolphosphate dehydrogenase-triosephosphate isomerase system;7 under assay conditions no indole appears in the reaction mixture, ensuring that reaction 2 activity is not being measured. Serine deamination is measured by observing pyruvate production with a coupled lactic dehydro- genase system. The reaction mixture consists of 0.1 M L-serine, 0.1 M Tris buffer, pH 7.8, 2 mM 390 Downloaded by guest on September 25, 2021 VOL. 51, 1964 BIOCHEMISTRY: CRAWFORD AND ITO 391 ammonium citrate, 1 mM P-mercaptoethanol, 0.1 mM DPNH, 28 .M pyridoxal phosphate, at least 80 units of lactic dehydrogenase (Type II, Nutritional Biochemicals), and enzyme. All reactions were performed at 37°. A unit of activity in any reaction is the disappearance of 0.1 umole of substrate or the appearance of 0.1 ismole of product in 20 min. Protein was deter- mined by the Lowry method.8 Specific activity refers to units of activity per milligram protein. Chemicals: Indoleglycerolphosphate was prepared by Yanofsky's method and purified by column chromatography.6 Results.-Demonstration of the reaction: Purified preparations of the B protein do not demonstrate DPNH oxidase activity. Such preparations catalyze L- serine-dependent DPNH oxidation in the coupled lactic dehydrogenase system described above. The amount of DPNH oxidation observed was proportional to time and B protein concentration in the standard assay mixture. Chroma- tography of the 2,4-dinitrophenylhydrazone of the keto acid formed in the reaction mixture confirmed its identity with pyruvic acid. Effect ofions: Reaction 3 catalyzed by isolated B protein proceeds at an optimal rate only at high ammonium ion concentrations.3 Figure 1 shows the effect of ammonium, potassium, and sodium ions on the L-serine deaminating activity of a given amount of purified B protein. 100 l Illll bc a. E N 0 20 _ 0.1 0.2 0.4 0.6 0.8 1.0 I.2 MOLARITY of CATION FIG. 1.-erine deaminase activity of normal B protein as a function of the cation present. Chromatographically purified component B was dialyzed against 0.1 M Tris-citrate buffer, pH 7.8, containing 1 mM i-mercaptoethanlol and 26 MM pyridoxal phosphate. Ammonium citrate was omitted from the usual reaction mixture; cations were added as citrates at pH 7.8. Pyridoxal phosphate requirement: B protein may be prepared in the apoenzyme form by the method of Matsuo and Greenberg,9 involving successive dialyses against 0.2 M Tris-chloride buffer and 0.2 M potassium phosphate buffer, both buffers at pH 7.8 and containing 1 mM,B-mercaptoethanol. When purified com- ponent B was resolved in this way, the activities shown in reactions 3 and 4 (with pyridoxal phosphate omitted from the assay mixtures) were found to be less than 5 per cent of those of the unresolved enzyme. Full activity in both reactions was regained immediately upon incubation with 28 jAM pyridoxal phosphate. Optimal pH: The maximal rate of L-serine deamination in standard assay mixtures occurred in the pH range 7.8-8.2. This corresponds to the pH optimum Downloaded by guest on September 25, 2021 392 BIOCHEMISTRY: CRAWFORD AND ITO PROC. N. A. S. 30 of the isolated B component acting in reaction 3 and the A-B complex acting in reactions 1 and Qt /3 as well. Km for L-serine: A Lineweaver-Burk plot of the relationship between reaction rate and L- serine concentration is shown as the lowest line 0 \ * in Figure 3. The Km for L-serine calculated 0 from these data is 1.45 X 102 M. Inhibition by indole: Purified component B /. thus appears capable of converting L-serine to two products, L-tryptophan or pyruvate, de- 0l 02 03 04 05 pending on the presence or absence of indole. indole pM It was first determined that both of these re- FIG. 2.-Relative reaction rates actions could be catalyzed simultaneously. The for pyruvate and tryptophan forma tion from L-serine as a function of inhibition caused by adding increasing amounts the initial indole concentration. of indole to a standard L-serine deamination as- Chromatographically purified nor- mal B protein was the catalyst in say containing 1.7 units of deaminating activity the usual serine deaminase reaction is shown in Figure 2. The amount of indole re- mixture. *, serine deamination, A, tryptophan formation. maining in the cuvettes after 20 min was deter- mined, and from this the rate of reaction 3 was estimated. From Figure 2, the Km for indole in reaction 3 was calculated to be 3 X 10-4 M, which is in good agreement with reported values for the indole Km using the isolated B protein. Figure 3 shows the result of an experiment to determine whether the inhibition of L-serine deamination brought about by indole was competitive or not. The lines do not intersect either at the ordinate (competitive inhibition) or the abscissa (noncompetitive inhibition). Under these circumstances the Ki for indole in this reaction may not be unequivocally determinable, but by employing a method recommended by Dixon and Webb,'" a value of 2.2 X 10-4 M was obtained. Effect of the A protein: The effect of adding A protein to B protein performing reaction 3 in dilute Tris or phosphate buffers is to increase the reaction rate 10- fold or more. Surprisingly, however, addition of A protein almost completely inhibits L-serine deaminase activity. When several amounts of dialyzed, crystalline A protein were added to cuvettes in which L-serine deamination was taking place, a new reaction rate was attained within 4 min. The new rate could be accounted for by the amount of B protein remaining uncomplexed. Again a series of L-serine saturation curves obtained at several levels of inhibition by the A protein failed to demonstrate clearly either its competitive or noncompetitive nature (Fig. 4). Boiled preparations of the A protein had no effect on the rate of L-serine deamination. Similarly, indoleglycerolphosphate and L-tryptophan were ineffective. Substrate specificity: Purified preparations of the B protein also deaminate L-threonine satisfactorily in assay mixtures containing this amino acid instead of L-serine. For these experiments, additional lactic dehydrogenase was added to allow for the slower rate of oxidation of a-ketobutyrate to a-hydroxybutyrate. At comparable substrate concentrations the reaction velocity with L-threonine was only 3 per cent of that attained with L-serine. This reaction rate was the Downloaded by guest on September 25, 2021 VOL. 51, 1964 BIOCHEMISTRY: CRAWFORD AND ITO 393 60 50- 40- 30- 2-0 I.0 1 -50 0 50 '100 150 /Es] FIG. 3.-The inhibition by indole of serine deamination catalyzed by normal B protein. o Lineweaver-Burk plot of reaction rate versus L-serine concentration with no indole present; v is expressed in serine deaminase units per 0.002 ml of B protein solution. 0, E0, U, and A are the same in the presence of 33 pM, 83 AM, 167 ;sM, and 500 ;MM indole.
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