IN the BIOSYNTHESIS of ANTHRANILATE by Americo RIVERA, JR.,* and P

864 BIOCHEMISTRY: RIVERA AND SRINIVASAN PROC. N. A. S. Summary.-By arbitrarily assuming one triplet code sequence for a single amino acid, it was possible to assign related base sequences for all other amino acids on the basis that a point mutation represents a single base change in the triplet. There are, therefore, six possible sets of codes and one of these six must represent the correct coding in template or messenger ribonucleic acid. The code inter- relationships indicate that, in a mutant, an amino acid is much more likely to be replaced by one with a different type of side chain (nonconservative) than by one having the same kind of side chain (conservative). * Aided by grants from the National Institutes of Health, U.S. Public Health Service. 1 Smith, E. L., these PROCEEDINGS, 48, 677 (1962). 2 Martin, R. G., J. H. Matthaei, 0. W. Jones, and M. W. Nirenberg, Biochem. Biophys., Re- search Commuz,., 6, 410 (1962). 3 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 441 (1962). 4 Abbreviations: U, A, G, and C are used for the nucleotide residues in ribonucleic acid, uridy- lic, adenylic, guanylic, and cytiylic, respectively. Amino acid residues are given in standard abbreviations. 6 References to amino acid substitutions have been given earlier.' 6 Henning, 1J., and C. Yanofsky, these PROCEEDINGS, 48, 183 (1962). 7Gordon, W. G., J. J. Basch, and E. B. Kalan, J. Biol. Chem., 236, 2908 (1961). 8 Piez, K. A., E. W. Davie, J. E. Folk, and J. A. Gladner, J. Biol. Chem., 236, 2912 (1961). 9 Kalan, E. B., W. G. Gordon, J. J. Basch, and R. Townend, Arch. Biochem. Biophys., 96, 376 (1962). 10 Base triplets in quotation marks refer to composition; sequence is not implied. 3-ENOIPYRUVYLSHIKIMATE 5-PHOSPHATE, AN INTERMEDIATE IN THE BIOSYNTHESIS OF ANTHRANILATE BY AMERICo RIVERA, JR.,* AND P. R. SRINIVASAN DEPARTMENT OF BIOCHEMISTRY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY Communicated by David Rittenberg, March 9, 1962 Cell-free extracts of Escherichia coli mutant B-37 can form anthranilate from shikimate 5-phosphate and L-glutamine.1 Mutants which accumulate an enolpyruvyl ether of shikimate (Zi) in their medium require the supplementation of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, and p-hydroxybenzoic acid for growth.2 This acid labile derivative of shikimate has been shown to be de- rived by a condensation of shikimate 5-phosphate and phosphoenolpyruvate in cell- free extracts.3 This has led to the suggestion that Z1 may be an intermediate between shikimate 5-phosphate and anthranilate.4 However, the cell-free system which forms anthranilate from shikimate 5-phosphate and L-glutamine cannot utilize Z1 as a carbon source.1 Recent structural studies indicate that ZI is 3-enolpyruvylshikimate.5' 6 Levin and Sprinson6 have reinvestigated the enzymatic condensation of shikimate 5-phosphate and phosphoenolpyruvate and demonstrated that these two components react to give 3-enolpyruvylshikimate 5-phosphate which is then dephosphorylated to yield 3-enolpyruvylshikimate (Zi). These new findings raise the interesting possibility that 3-enolpyruvylshikimate 5-phosphate and not its dephosphorylated derivative, 3-enolpyruvylshikimate, is the real intermediate. This Downloaded by guest on September 25, 2021 VOL. 48, 1962 BIOCHEMISTRY: RIVERA AND SRINIVASAN 865 TABLE 1 SUBSTRATE REQUIREMENTS FOR THE FORMATION OF ANTHRANILATE WITH THE PURIFIED ENZYME FRACTIONS OF E. coli B-37 Yield of anthranilate Additions (pmoles) None 0 Shikimate 5-phosphate 0.01 Shikimate 5-phosphate + phosphoenolpyruvate 0.25 Shikimate.5-phosphate + pyruvate 0.01 3-Enolpyruvylshikimate 5-phosphate 0.24 The reaction mixture contained 0.5 jmole of glutamine, 5 jtmoles of Mg + +, 5 emoles of reduced glutathione, 50 gmoles of Tris buffer pH 8.2, 0.5 smole of DPN, 150 Mmoles of alcohol, 0.05 ml of alcohol dehydrogenase (3 mg/ml), 0.2 ml of fractionated enzyme (2.36 mg of protein), and 0.5 ,&mole of the indicated substrates in a total volume of 1 ml. After incubation at 370 for 2 hr the reaction was terminated by the addition of 0.4 ml of trichloracetic acid (10 per cent), centrifuged and the anthranilic acid formed was determined on aliquots of the supernatant. communication is concerned with the role of 3-enolpyruvylshikimate 5-phosphate in the biosynthesis of anthranilic acid. Cell-free extracts of E. coli B-37, Aerobacter aerogenes mutants A170-40 and A170- 44 were prepared as described in an earlier publication.1 E. coli B-37 accumulates anthranilate and is blocked immediately after it.7 A170-40 and A170-44 are quin- tuple auxotrophs which require the supplementation of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, and p-hydroxybenzoic acid for growth.2 A170-40 accumulates shikimate 5-phosphate in the medium and is blocked between shikimate 5-phosphate and 3-enolpyruvylshikimate 5-phosphate. A170-44 accumulates 3-enolpyruvylshikimate and a little 3-enolpyruvylshikimate 5-phosphate and is blocked immediately after the latter compound. Anthranilic acid was determined on aliquots of trichloracetic acid treated supernatants by the method of Bratton and Marshall8 as modified by Eckert.9 Since 3-enolpyruvylshikimate 5-phosphate is formed by a condensation of shikimate 5-phosphate and phosphoenolpyruvate, cell-free extracts of B-37 were investi- gated for stimulation of anthranilate synthesis by phosphoenolpyruvate in the presence of shikimate 5-phosphate and minimal amounts of glutamine. With phosphoenolpyruvate neither the rate of synthesis of anthranilate nor the total amount of anthranilate formed was altered appreciably.10 Attempts to demonstrate the direct conversion of 3-enolpyruvylshikimate 5-phosphate to anthranilate with cell-free extracts of B-37 were also inconclusive due to the rapid conversion of 3-enolpyruvylshikimate 5-phosphate to shikimate 5-phosphate in these extracts. In order to sur- mount these difficulties the cell-free extracts of B-37 were fractionated once with protamine and twice with (NH4)2SO4. Enzyme fractions thus obtained were com- pletely incapable of converting shikimate 5-phosphate to anthranilate in the presence of glutamine and DPN. However, the addition of phosphoenolpyruvate, reduced glutathione and a regenerating system for DPNH (or TPNH) restored the synthesis (Table 1). Phosphoenolpyruvate cannot be replaced by pyruvate. Shikimate 5-phosphate and phosphoenolpyruvate can be replaced by their enzymatic condensation product 3-enolpyruvylshikimate 5-phosphate. These results suggest that in the fractionated enzyme system shikimate 5-phosphate and phosphoenolpyruvate condense to yield 3-enolpyruvylshikimate 5-phosphate which is then converted to anthranilate in the presence of glutamine (Fig. 1). Further confirmation for the role of 3-enolpyruvylshikimate 5-phosphate in the Downloaded by guest on September 25, 2021 866 BIOCHEMISTRY: RIVERA AND SRINIVASAN PROC. N. A. S. COOH COOH POFIII$IIXO + COOH I1 COOH PO", OH IH PO' ° OH 2 OH CH2 Shikimote Phosphoenol- 3-Enolpyruvylshikimote 5-Phosphate pyruvate 5-Phosphote COOH CONH2 OOOH P0 t.oQ H-C-NH2 CH 11H OH2 COOH 3-Enolpyruvyl- L-Glutomine Anthronilic shikimote Acid 5-Phosphate FIG. 1 biosynthesis of anthranilate was obtained by comparing the behavior of extracts of A170-40 and A170-44 toward different substrates (Table 2). Extracts of A170-44 synthesize negligible amounts of anthranilate from either shikimate 5-phosphate plus phosphoenolpyruvate and glutamine or 3-enolpyruvylshikimate 5-phosphate and glutamine. This is in agreement with the mutational locus assigned to this mutant from growth requirements and accumulation studies, i.e., A170-44 is blocked immediately after 3-enolpyruvylshikimate 5-phosphate and is unable to convert it to intermediates capable of participating in the biosynthesis of aromatic amino acids. TABLE 2 BEHAVIOR OF EXTRACTS OF A170-40 AND A170-44 TOWARD VARIOUS SUBSTRATES Enzyme Yield of extract anthranilate Substrates used (1sMoles) Shikimate 5-phosphate + phosphoenolpyruvate A170-40 0.01 Shikimate 5-phosphate + phosphoenolpyruvate A170-44 0.01 3-Enolpyruvylshikimate 5-phosphate A170-40 0.31 3-Enolpyruvylshikimate 5-phosphate A170-44 0.03 The reaction mixture contained 2.5 Moles of glutaimine, 50 sAmoles of Tris buffer pH 8.2, 5 /Amoles of Mg + +, 5 wsmoles of reduced glutathione, 0.5 smole of DPN, 150 jsmoles of alcohol, 0.05 ml of alcohol dehydrogenase (3 mg/ml), 0.1 ml of enzyme extract (20 mg of protein per ml), and 0.5 jomole of the indicated substrates in a total volume of 1 ml. After incubation at 370 for 2 hr. the reaction was stopped with 0.4 ml of trichloracetic acid (10%), and centrifuged, and the anthranilic acid formed was estimated on aliquots of the supernatant. Unlike A170-44, extracts of A170-40 can form anthranilate from 3-enolpyruvylshikimate 5-phosphate and glutamine. However, shikimate 5-phosphate and phosphoenolpyruvate cannot substitute for 3-enolpyruvylshikimate 5-phosphate, thus confirming the absence of the condensing enzyme for 3-enolpyruvylshikimate 5-phosphate in these extracts. These enzyme studies with the mutant extracts support the conclusion that 3-enolpyruvylshikimate 5-phosphate is an intermediate in the biosynthesis of anthranilate. Recently we have shown that p-aminobenzoate is also synthesized from shikimate 5-phosphate and L-glutamine ill cell-free extracts of baker's yeast.11 Mutants which accumulate 3-enolpyruvylshikimate require not only the addition of phenylalanine, tyrosine and tryptophan but also p-aminobenzoic acid in their medium for growth.2 The existence of these mutants suggests that 3-enolpyruvylshikimate Downloaded by guest on September 25, 2021 VOL. 48, 1962 BIOCHEMISTRY: E. STEERS, JR. 867 5-phosphate may also participate as an intermediate in the biosynthesis of p-aminobenzoate. Investigations are in progress to examine this possibility. This work was supported by a research grant from the National Institutes of Health, U.S. Public Health Service, R.G.

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