Nonidentity of the Aspartate and the Aromatic Aminotransferase Components of Transaminase A in Escherichia colil

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1 JOURNAL OF BACrERIOLOGY, OCt. 1972, p Copyright i 1972 American Society for Microbiology Vol. 112, No. 1 Printed in U.S.A. Nonidentity of the Aspartate and the Aromatic Aminotransferase Components of Transaminase A in Escherichia colil R. H. COLLIER AND G. KOHLHAW Department of Biochemistry, Purdue University, West Lafayette, Indiana Received for publication 15 May 1972 Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000. In 1953, Rudman and Meister demonstrated that there exist at least three distinct aminotransferases in Escherichia coli (13). The first, designated "transaminase A," was shown to catalyze transamination between glutamate and aspartate, tryptophan, tyrosine, and phenylalanine, as well as, to a lesser extent, methionine and leucine. The second, "transaminase B," catalyzed transamination between glutamate and leucine, isoleucine, and valine, exhibiting essentially no activity toward aspartate, tryptophan, and tyrosine and little activity toward methionine and phenylalanine. The third enzyme, later termed "transaminase C" (11), was active with valine, alanine, and a- aminobutyrate. The A-B nomenclature has since commonly been employed in the literature, and to our knowledge no further fractionation has been reported, although a number of additional E. coli aminotransferase activities have been described (e.g., 7, 12, 19, 20). Repression of transaminase A by tyrosine was reported by Silbert, Jorgensen, and Lin (16), and the gene controlling this repression was later designated tyrr by Wallace and Pittard (21). If indeed transaminase A was one protein exhibiting all the activities mentioned above, then its repressibility by tyrosine would constitute a peculiar situation, particularly in view of the probable involvement of aspartate aminotransferase activity in the biosynthesis of a large number of metabolites. We have investigated the identity of the aspartate and aromatic aminotransferase activities of transaminase A, and we conclude from the experiments to be presented here that the aspartate activity resides in a separate protein. A brief account of our results has been presented (R. H. Collier, Fed. Proc. 31:497, 1972). MATERIALS AND METHODS most experi- I Journal paper no of the Purdue University Agricultural Experiment Station. ments was E. coli CU2 (K-12), a transaminase B Organisms. The organism used in 365

2 366 COLLIER AND KOHLHAW J. BACTERIOL. mutant, kindly provided by H. E. Umbarger, Purdue University. This strain was previously known as 11 A 16 (18). Some growth properties pertaining to this investigation are given in the Results section. E. coli CS8TA (K-12), a mutant with a low aspartate aminotransferase level (9), as well as its parent, CS8, were obtained from Y. S. Halpem, Hebrew University, Jerusalem. CS8TA had been selected from CS8 as one of several mutants which had lost the ability to grow on glutamate as the sole carbon source. Both CS8 and CS8TA also require methionine for growth (9) Ċhemicals. Special chemicals and their suppliers: L-aspartic acid, L-glutamic acid, L-tyrosine, L-phenylalanine, L-tryptophan, L-leucine, L-isoleucine, L- valine, L-methionine, a-ketoglutaric acid, a-ketoisovalerate (sodium), phenazine methosulfate, nitroblue tetrazolium, and L-glutamic dehydrogenase (type II) were purchased from Sigma. Oxalacetic acid was obtained from Calbiochem. Pyridoxal-5'-phosphate (PLP) was a product of Pierce Biochemicals. Tris- (hydroxymethyl)aminomethane (Tris), special enzyme grade, was purchased from Schwarz-Mann. Growth media. The basal medium was the Davis and Mingioli medium (4), modified by omitting citrate and increasing the glucose concentration to 0.5%. Amino acid supplements were made as indicated in the text. Cells were grown aerobically at 37 C in 1-liter batches and harvested at the end of log phase. Preparation of crude extracts. One part of cells (wet weight) was suspended in four parts of cold 0.1 M potassium phosphate buffer, ph 7.4, and passed through a French pressure cell (Aminco) at 1,000 to 1,200 psi. The resulting suspension was immediately cooled in an ice bath and centrifuged for 1 hr at 40,000 x g. The supernatant solution constituted the crude extract. Its protein concentration was usually about 12 mg/ml, as determined with the biuret reaction. Enzyme assays. Aspartate aminotransferase activity (EC ) was assayed by measuring the appearance of oxaloacetate, indicated by an increase in optical density (OD) at 265 nm (6). The assay mixture contained, in 1.0 ml: 200,umoles of Trishydrochloride buffer, ph 8.0, 40 gmoles of L-aspartate, 40 umoles of a-ketoglutarate, and 0.1 Amole of PLP. The reaction was initiated with enzyme, and the increase in OD was followed using a Gilford 240 spectrophotometer and a Sargent SRLG recorder. The temperature was 25 C. A molar extinction coefficient of 780 M-1 cm-' was employed. Specific activity is defined as micromoles of oxaloacetate formed per minute per milligram of protein. Tyrosine aminotransferase activity (EC ) was measured by estimating the appearance of p- hydroxyphenylpyruvate. The reaction mixture contained, in 0.5 ml: 50,moles of potassium phosphate buffer, ph 7.4, 1,umole of L-tyrosine, 10 Asmoles of a- ketoglutarate, and 0.1 Amole of PLP. The reaction was initiated with enzyme. After 10 min at 37 C, 0.5 ml of 2 N NaOH was added. The alkaline solution was incubated for 30 min at room temperature. Then the OD at 330 nm was read against a control to which NaOH had been added at 0 min. The molar extinction coefficient of p-hydroxyphenylpyruvate at 330 nm was determined to be 19,500 M- I cm- I under the condition of the assay. Specific activity is defined as micromoles of p-hydroxyphenylpyruvate formed per minute per milligram of protein. Tryptophan aminotransferase activity was assayed by measuring the appearance of indole pyruvate. The conditions were exactly as described for the tyrosine aminotransferase except that L-tryptophan was substituted for L-tyrosine. The molar extinction coefficient of indole pyruvate at 330 nm was determined to be 10,000 M- I cm- ' under assay conditions. Specific activity is defined as micromoles of indole pyruvate formed per minute per milligram of protein. The measurement of phenylalanine aminotransferase activity again followed that of tyrosine aminotransferase except that the assay mixture contained 2.5 Mmoles of L-phenylalanine and that the formation of phenylpyruvate was analyzed at 320 nm. The OD was read immediately after addition of NaOH. A molar extinction coefficient of 17,500 M- I cnm- ' was used (3). Specific activity is defined as micromoles of phenylpyruvate formed per minute per milligram of protein. Glutamate-a-ketoisovalerate transamination served as a measure for transaminase B activity (EC e.). The assay was coupled to glutamic dehydrogenase. The reaction mixture contained, in 1.0 ml: 200,moles of Tris-hydrochloride buffer, ph 8.0, 40 qmoles of L-glutamate, 4,umoles of a-ketoisovalerate, 40 umoles of NH4Cl, excess glutamic dehydrogenase (0.1 unit), 0.25 umoles of PLP, 0.2 mg of reduced nicotinamide adenine dinucleotide (NADH), and 1 Mmole of NaNs to inhibit NADH oxidase. After addition of enzyme-containing sample, the decrease in OD at 340 nm was observed. Temperature was 25 C. Values were corrected for blank reaction in absence of a-ketoisovalerate. Specific activity is expressed as micromoles of NADH oxidized per minute per milligram of protein. Disc gel electrophoresis. The procedure of Davis (5) was followed except that spacer and sample gel were omitted. The samples were applied in 10% (v/v) glycerol to gels which had been previously subjected to electrophoresis for approximately 1 hr. Electrophoresis proper was performed for 90 min at 2 ma per gel and at 5 C. Thereafter, gels were stained for aminotransferase activity by the method of Ryan et al. (14). This method is based on the formation of glutamate from a-ketoglutarate and the desired amino acid substrate; redox reactions involving NAD+, phenazine methosulfate, and nitro blue tetrazolium subsequently lead to the formation of purple reduced nitro blue tetrazolium. The general incubation mixture contained, in 30 ml: a-ketoglutarate, 30 mg; NAD+, 60 mg; phenazine methosulfate, 1 mg; nitro blue tetrazolium, 15 mg; glutamic dehydrogenase, 2.9 units; PLP, 2 mg; NaN, 20 mg; and 0.1 M K-phosphate buffer, ph 7.5. To this was added the desired amino acid at a concentration comparable to that used in the normal activity assay. No color appeared during the incubation period of 45 min when no amino acid was added. Other control gels were routinely included which were stained for protein, using amidoschwarz.

3 VOL. 112, 1972 AMINOTRANSFERASE COMPONENTS OF TRANSAMINASE A RESULTS Influence of various amino acids on the growth of strain CU2. Most experiments described were performed with extracts from transaminase B-less strain CU2. Owing to the absence of active transaminase B, this organism requires isoleucine for growth, but not leucine or valine. This was expected, since transaminase A exhibits some activity toward leucine and since valine can be produced by transamination between a-ketoisovalerate and alanine or a-aminobutyrate (1, 13). It appeared, however, that leucine transamination was rate limiting when isoleucine was the only supplement. As shown in Fig. 1, the mass doubling time in log phase decreased from about 200 min in the presence of isoleucine to 100 min in the presence of both isoleucine and leucine (curves 1 and 2, Fig. 1). Inclusion of valine had no further effect. The doubling time of 100 min was comparable to that of the parent strain E. coli K-12, grown under similar conditions (17). When tyrosine, which is known to 0 In INCUBATION TIME, HOURS FIG. 1. Log-phase growth of E. coli CU2. The minimal glucose medium was supplemented as follows, with all amino acids being added at an initial concentration of 1 mm: 1, isoleucine + leucine + (valine); 2, isoleucine + (aspartate); 3, isoleucine + tyrosine + leucine + (phenylalanine); 4, isoleucine + tyrosine + (aspartate). The amino acids listed in parentheses could be omitted without a change in growth rate. In all instances, 0.2 ml of an inoculum, grown in nutrient broth, was added to 1 liter of growth medium. Cultures were grown with shaking at 37 C. The stationary phase was reached at an OD540 of 1.6 to 1.7. partially repress the tyrosine and (heat-labile) phenylalanine aminotransferase activities (16), was added to the medium, the growth rate decreased four- to fivefold (curve 4, Fig. 1). This effect could be largely reversed by the addition of leucine. However, growth on isoleucine, tyrosine, and leucine was still significantly slower than growth on isoleucine and leucine (curves 1 and 3 of Fig. 1). The most likely reason for this could be a requirement for phenylalanine, since the specific (heat-stable, nonrepressible) phenylalanine aminotransferase comprises no more than one-fifth of the total phenylalanine aminotransferase (16). If a requirement for phenylalanine existed, then addition of this amino acid to the medium should have increased the growth rate. The failure of phenylalanine to do so in our experiments (curve 3, Fig. 1) is inconclusive. It could reflect a competition of tyrosine and phenylalanine for entrance into the cell (2). Aspartate, which has also been considered to be a substrate of transaminase A had no effect on the growth rate of strain CU2 under the conditions tested (Fig. 1). Specificity of repression of aminotransferase activities by tyrosine. Table 1 lists relative activities of various aminotransferases in cells grown with tyrosine, phenylalanine, tryptophan, aspartate, or tyrosine plus aspartate in addition to the three branched-chain amino acids. It is evident that tyrosine re- TABLE 1. Specificity of repression of aminotransferase activities of E. coli CU2 by tyrosine Relative activities of aminotransferases Growth supplements" Aspar- Tyro- Pylhaelan- Tryptate sine nine tophan 367 None " Tyrosine, 1 mm Phenylalanine mM Tryptophan, mm Asparate, 1 mm Tyrosine, 1 mm aspartate, 1 mm I: I I_ ain addition to 1 mm each of isoleucine, valine, and leucine. "The actual specific activities under assay conditions (see Materials and Methods) were: aspartate aminotransferase, 0.73; tyrosine aminotransferase, 0.23; phenylalanine aminotransferase, 0.20; tryptophan aminotransferase, 0.28; valine aminotransferase (transaminase B), <0.001.

4 368 COLLIER AND KOHLHAW J. BACTERIOL. pressed the activities of tyrosine aminotransferase and of tryptophan aminotransferase by about 80%, whereas the phenylalanine aminotransferase activity was lowered by about 65%, confirming results obtained previously (16). The interestiiig point, however, is that the aspartate arninotransferase level was not changed by tyrosine (nor by 15 other amino acids tested). Since this suggested that the tyrosine and the aspartate aminotransferase activities might be separate enzymes, further experiments were initiated to check this possibility. Heat sensitivity of aspartate, tyrosine, and phenylalanine aminotransferase activities. When kept at 55 C under the conditions described in the legend of Fig. 2, only 20% of the aspartate aminotransferase was inactivated in 1 hr whereas the phenylalanine and tyrosine aminotransferase activities decreased by 84 and 93%, respectively. The stability of the aspartate aminotransferase was noteworthy, especially since no substrates were present, and contrasted sharply with the lability of the phenylalanine and tyrosine activities. The slightly greater stability of the phenylalanine aminotransferase may be explained by the presence of the heat-stable form of this enzyme described previously (16). Apoenzyme formation. Further evidence for the nonidentity of the aspartate and tyrosine aminotransferases came from experiments de- signed to study the rate of apoenzyme formation (Fig. 3). The curves reflect the amounts of apoenzyme present at any given moment over an 8-hr period. The failure to obtain all enzyme in the apo form is, we believe, due to apoenzyme instability. Therefore, each point actually represents a composite measure of formation and breakdown of the apoenzymes. It appears, however, that apoenzyme formation is absent in the final stages of the experiment. One may therefore conclude that at least the tates of apoenzyme breakdown are different, with the aspartate aminotransferase apoenzyme being much more stable than the tyrosine aminotransferase apoenzyme. The degradation of both apoenzymes appeared to be first order (Fig. 3), with rate constants of hr- ' and hr-1 for the aspartate aminotransferase and the tyrosine aminotransferase, respectively. The half-lives, calculated from these rate constants, were 13.0 and 3.6 hr, respectively. Under the conditions listed in Fig A SP A.T. / _ Downloaded from > TIME AT 55, MIN. FIG. 2. Heat stability of aspartate, tyrosine, and phenylalanine aminotransferase (ASP A.T., TYR A.T., and PHE A.T., respectively) activities of E. coli CU2. Crude extract, prepared as described in Materials and Methods, was passed through Sephadex G-25. (This procedure did not lead to formation of apoenzyme.) The protein-containing fractions were combined and the pool was used directly in the heat stability experiment. Incubation was in 0.1 M Tris-hydrochloride buffer, ph 8.0. TIME OF DIALYSIS, HOURS FIG. 3. Formation by dialysis of the apoenzyme forms of aspartate and tyrosine aminotransferases of E. coli CU2. Material obtained from Sephadex G- 150 filtration (cf. Fig. 5) was dialyzed against 1,000 volumes of 0.1 M Tris-hydrochioride buffer, ph 8.0 at 4 C. At the indicated times, activities were measured in the absence and in the presence of pyridoxal- 5-phosphate (PLP) (0.2 mm). Enzyme and PLP were previously incubated for 10 mm to allow complete recombination. The difference between the two measurements, representing reactivable apoenzyme, was plotted on a percentage scale, with 100% reflecting the theoretically possible apoenzyme concentration, which is identical to the concentration of holoenzyme at the start of the experiment. A firstorder plot for apoenzyme degradation is also shown. a, Concentration of apoenzyme at 5.5 hr; a - x, concentration of apoenzyme remaining after time t; 0, aspartate aminotransferase; 0, tyrosine aminotransferase. on September 22, 2018 by guest

5 VOL. 112, 1972 AMINOTRANSFERASE COMPONENTS OF TRANSAMINASE A 3, the two holoenzymes were completely stable. Tyrosine aminotransferase level in an aspartate aminotransferase mutant. In their work on glutamate metabolism in E. coli K- 12, Marcus and Halpern described a mutant, CS8TA, which exhibited only 10% of the aspartate aminotransferase activity of the parent, CS8 (9). It was of interest with respect to the present study to find out whether or not the tyrosine aminotransferase of the mutant had also been altered. Table 2 shows that this was not the case: the tyrosine aminotransferase activities were identical in both strains, whereas the aspartate aminotransferase activities showed the expected difference. Disc gel electrophoresis of crude extracts. Gel electrophoresis with subsequent activity staining clearly established the nonidentity of the aromatic and aspartate aminotransferases of E. coli and confirmed the notion that the tyrosine, phenylalanine, and tryptophan aminotransferase activities are likely to be carried by the same protein. The results have been schematized in Fig. 4. It is evident that the ratio of the various activities as measured by the intensity of color after 45 min differs from the ratio of the specific activities listed in Table 1. The reasons for this apparent discrepancy are not clear. It should be noted, however, that the activities observed on the gels do not necessarily represent initial velocities. Also, the assay conditions were not the same in the gel staining procedure and in the determination of specific activities. Finally, the influence of the conditions of electrophoresis on individual enzyme stability is not known. The somewhat broad and diffuse phenylalanine aminotransferase band may reflect similar but nonidentical mobilities of the heat-labile and heat-stable forms of this enzyme. In one experiment, this region was actually resolved into two separate bands. Gel filtration experiments. Crude extracts were subjected to gel filtration on Sephadex G- TABLE 2. Specific activities of tyrosine and aspartate aminotransferase activities in crude extracts of E. coli CS8 and CS8TA Enzyme Specific activity (Amoles per min per mg of protein) CS8 CS8TA Tyrosine aminotransferase Aspartate aminotransferase in order to explore the usefulness of such treatment for the separation and purification of the aromatic and aspartate aminotransferases, and also to estimate molecular weights. The large-scale experiment shown in Fig. 5 indicated a great similarity in size of the tyrosine (or phenylalanine) and aspartate aminotransferases. The slightly stronger retardation of the aspartate aminotransferase relative to the other two activities was reproducible and may reflect a difference in shape rather than in size. On a smaller analytical column of Sephadex G-150 calibrated with ribonuclease, chymotrypsinogen A, ovalbumin, and aldolase, the molecular weights of both the aspartate and the tyrosine aminotransferase were estimated to be close to 100,000. It is noteworthy that comparable values have been reported for aspartate aminotransferases from a variety of sources, including Rhizobium japonicum (14), yeast (15), soybeans (14), and ox heart (10). ORIGIN > TRACKING _ DYE TYR -l_ PHE n TRP e ASP 369 ASP + TYR FIG. 4. Disc gel electrophoresis of crude extract of E. coli CU2. Electrophoresis was performed as described in Materials and Methods. The samples applied (0.05 ml were 40 mm in Tris-hydrochloride buffer, ph 8.0, and contained 240,gg of protein and 10% (v/v) glycerol. After electrophoresis, the gels were incubated with activity stain (see Materials and Methods) and left in the dark for 45 min (room temperature). Thereafter, the bands shown in the figure were clearly visible as purple rings. The gels were then scanned at 640 nm using the "linear transport" attachment of the Gilford model 240 spectrophotometer. The relative intensities of the bands were (with the R, values of the centers of the bands given in parentheses): tyrosine (TYR) aminotransferase, 100 (0.51); phenylalanine (PHE) aminotransferase, 31 (0.49); tryptophan (TRP) aminotransferase, 88 (0.51); aspartate (ASP) aminotransferase, 15 (0.40).

6 370 COLLIER AND KOHLHAW J. BACTERIOL PROTEIN ASP A.T : c m E 4z FG-f4G TYR~~~~~~~~~~~~ A.T. -z PIE AT ELUATE, ml. FIG. 5. Gel filtration of crude extr-act of E. coli CU2 on Sephadex G-150 equilibrated with 0.2 M Tris-hydrochloride buffer, ph 8.0, containing 10 p,m pyridoxal-5'-phosphate. A 25-ml amount of extract was applied to a column of 1,800-mi total volume (diameter 5 cm, height 92 cm). Using upward flow, a flow rate of 72 mi/hr was obtained. Fractions of 12 ml were collected and the protein concentration as well as the aspartate, tyrosine, and phenylalanine aminotransferase activities were determined. Activities are expressed as micromoles of product formed per hour per milliliter. U.W U) _j DISCUSSION Our results strongly suggest that the classical transaminase A of E. coli consists of at least two different enzymes, one of which is reactive with glutamate and aspartate and another which interacts with glutamate and the aromatic amino acids (tyrosine, phenylalanine, tryptophan) and possibly also with methionine and leucine. The latter two amino acids produced the lowest rates with the transaminase A preparation of Rudman and Meister (13) and have not been investigated in the present work. It appears, however, that the leucine and the aromatic aminotransferase activities are corepressed by tyrosine, since the growth inhibition of strain CU2 by tyrosine could be largely reversed by leucine (see Fig. 1), suggesting that a leucine requirement had been created by the presence of tyrosine. The finding that the aspartate aminotransferase was not repressed by amino acids was not unexpected, since in the presence of glucose, where aspartase is low, aspartate aminotransferase is probably the only provider of aspartate, which in turn is the precursor both for several amino acids and for pyrimidine and purine nucleotides. The tryptophan and methionine aminotransferase activities deserve special attention since their physiological significance is not quite clear at present. The biosynthesis of neither amino acid involves transamination of the corresponding a-ketoacids. However, as was recently suggested by Kuhn and Somerville (8), those activities may have a part in the metabolism of D-tryptophan and D-methionine. It is hoped that more detailed studies of the substrate specificity of individual aminotransferases will shed more light on their relative importance. Purification of the aromatic and aspartate aminotransferases, prerequisite for meaningful kinetic investigations, is now in progress. ACKNOWLEDGMENTS We wish to thank H. E. Umbarger and Y. S. Halpern for providing us with the bacterial strains used in this study, and R. L. Somerville for critically reviewing the manuscript. We gratefully acknowledge the skillful assistance of R. Bohme in the gel electrophoresis experiments. This work was supported by a David Ross Research Grant from the Purdue Research Foundation (PRF-6245) and by Public Health Service grant no. GM from the National Institute of General Medical Sciences. LITERATURE CITED 1. Adelberg, E. A., and H. E. Umbarger Isoleucine and valine metabolism in Escherichia coli. a-ketoisovaleric acid accumulation. J. Biol. Chem. 205: Brown, K. D Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol. 104: Cotton, R. G. H., and F. Gibson The biosynthesis of phenylalanine and tyrosine; enzymes converting chorismic acid into prephenic acid and their relationships to prephenate dehydratase and prephenate dehydrogenase. Biochim. Biophys. Acta 100: Davis, B. D., and E. S. Mingioli Mutants of Esch-

7 VOL. 112, 1972 AMINOTRANSFERASE COMPONENTS OF TRANSAMINASE A 371 erichia coli requiring methionine or vitamin B2, J. Bacteriol. 60: Davis, B. J Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: Dixon, H. B. F., and E. S. Severin Dissociation of the prosthetic group of asparate amino transferase. Biochem. J. 110: 18P-19P. 7. Kim, K.-H., and T. T. Tchen Putrescine-a-ketoglutarate transaminase in Escherichia coli. Biochem. Biophys. Res. Commun. 9: Kuhn, J., and R. L. Somerville Mutant strains of Escherichia coli K12 that use D-amino acids. Proc. Nat. Acad. Sci. U.S.A. 68: Marcus, M., and Y. S. Halpern The metabolic pathway of glutamate in Escherichia coli K-12. Biochim. Biophys. Acta 177: Marino, G., A. M. Greco, V. Scardi, and R. Zito Purification and general properties of asparate aminotransferase of ox heart. Biochem. J. 99: Meister, A. (ed.) Biochemistry of the amino acids, 2nd ed., vol. 1, p Academic Press Inc., New York. 12. Peterkofsky, B., and C. Gilvarg N-Succinyl-Ldiaminopimelic-glutamic transaminase. J. Biol. Chem. 236: Rudman, D., and A. Meister Transamination in Escherichia coli. J. Biol. Chem. 200: Ryan, E., F. Bodley, and P. F. Fottrell Purification and characterization of aspartate aminotransferases from soybean root nodules and Rhizobium japonicum. Phytochemistry 11: Schreiber, G., M. Eckstein, G. Maas, and H. Holzer Die physikalisch-chemischen Eigenschaften von Apoaspartataminotransferase aus Bierhefe. Biochem. Z. 340: Silbert, D. F., S. E. Jorgensen, and E. C. C. Lin Repression of transaminase A by tyrosine in Escherichia coli. Biochim. Biophys. Acta 73: Temple, R. J., H. E. Umbarger, and B. Magasanik The effect of L-valine on enzyme synthesis in Escherichia coli K-12. J. Biol. Chem. 240: Umbarger, H. E., and J. H. Mueller Isoleucine and valine metabolism of Escherichia coli. I. Growth studies on amino acid-deficient mutants. J. Biol. Chem. 189: Umbarger, H. E., M. A. Umbarger, and P. M. L. Siu Biosynthesis of serine in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 85: Vogel, H. J., D. F. Bacon, and A. Baich Induction of acetylornithine 6-transaminase during pathwaywide repression, p In H. J. Vogel, V. Bryson, and J. 0. Lampen (ed.), Informational macromolecules. Academic Press Inc., New York. 21. Wallace, B. J., and J. Pittard Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli. J. Bacteriol. 97: Downloaded from on September 22, 2018 by guest