IV-Acetylglutamate Synthase of Escherichia coli Regulation of Synthesis and Activity by Arginine”

N-Acetylglutamate synthase, the first enzyme of arginine biosynthesis, was stabilized in crude extracts from Escherichia coli. At 4 degrees the enzyme lost less than 5% of activity per day. L-Arginine repressed the formation of N-acetylglutamate synthase. Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured. No activity (i.e. less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cell cultivated on minimal medium. In a 6-fold purified preparation L-arginine inhibited the enzyme. Of 11 precursors and analogues of arginine tested only O-[L-norvalyl-5]-isourea inhibited N-acetylglutamate synthase as strongly as L-agrinine.


IV-Acetylglutamate
At 4" the enzyme lost less than 5% of activity per day. L-Arginine repressed the formation of N-acetylglutamate synthase. Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured.
No activity (i.e. less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cells cultivated on minimal medium. In a 6-fold purified preparation L-arginine inhibited the enzyme. Of 11 precursors and analogues of arginine tested only O-[L-norvalyl-5]-isourea inhibited N-acetylglutamate synthase as strongly as L-arginine. N -Acetylglutamate synthase (acetyl-CoA: L glutamate Nacetyltransferase, EC 2.3.1. l), the first enzyme of arginine biosynthesis, catalyzes the acetyl-CoA-dependent acetylation of the amino group of glutamic acid. This reaction was first demonstrated in crude extracts of Escherichia coli by Maas et al. (1). Further studies on the N-acetylglutamate synthase from this organism have been hindered by the instability of the enzyme in crude extracts and by the lack of a specific in vitro assay. Vyas and Maas (2) therefore used resting cell suspensions as a source of enzyme and measured N-acetylglutamate formation in a bioassay. In the range of 1 to 25 nmol, the amount of "Pi formed was proportional to the amount of N-acetylglutamate. This sensitive and specific method for the determination of Nacetylglutamate is described in detail elsewhere.2

RESULTS
Stability of Enzyme-In cell extracts prepared as described under "Materials and Methods" N-acetylglutamate synthase was reasonably stable, losing less than 5y0 of its activity per day at 4". At room temperature the enzyme was unstable. When glycerol was omitted from the extraction buffer, the enzyme activity dropped rapidly after extraction and could not be detected after 12 hours at 4". Inclusion of the protease inhibitor phenylmethylsulfonyl fluoride (16) in the extraction buffer exerted a stabilizing effect on N-acetylglutamate synthase but was without effect when added at 200 mg per liter to the growth medium.
Assay-Under standard assay conditions the acetyl-CoA-dependent formation of labeled derivatives other than N-acetyl-[14C]glutamate from [r4C]glutamate cannot be excluded a priori.
However, evidence in support of N-acetylglutamate as the main labeled product formed was obtained when we detected approximately the same amount of N-acetylglutamate either by the standard assay or by a specific enzymatic assay using N-acetylglutamate 5-phosphotransferase.
In an incubated reaction mixture we determined 18.7 nmol of N-acetylglutamate by assaying the 14C radioactivity after treatment with Dowex 50-W (standard assay) and 21.1 nmol of N-acetylglutamate with the N-acetylglutamate 5-phosphotransferase reaction.
An additional in-dication for the specificity of the standard N-acetylglutamate assay was obtained with the argA-strain 39A23R3. This strain was grown under conditions leading to derepression of the arginine biosynthetic enzymes (Medium E with 16 pg of N2-acetylornithine per ml).
When an extract of these cells was assayed by the standard method we detected no formation of N-acetyl-[r4C]glutamate.
In the presence of 2 mM arginine, N-acetylglutamate synthase activity was drastically reduced but not completely inhibited. The low residual activity could not be reduced further by higher arginine concentrations.
Irrespective of the N-acetylglutamate assay used, it amounted to about 12% of the uninhibited activity. From this observation we conclude that the residual N-acetylglutamate synthase activity, observed in the presence of arginine, is not due to some interfering side reactions, The standard incubation conditions in the N-acetylglutamate synthase assay were chosen on the basis of the following considerations: between pH 8 and 9 enzyme activities were higher in Tris buffer than in phosphate buffer, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, sodium 5,5-diethylbarbiturate-HCl, or glycine-NaOH.
With Tris buffer, we observed a pH optimum at 9.0. Glycerol, which is essential for the stability of the enzyme in the extraction buffer, was included, at 15% (v/v), in the standard incubation mixture. It exerts a slight depression of the enzyme activity and alters the feedback properties of the enzyme (compare Fig. 2). Aminooxyacetic acid (7 mM) inhibits arginine decarboxylase (17) but has no influence on the N-acetylglutamate synthase activity. Under standard incubation conditions the concentrations of acetyl-CoA and ['"Clglutamate were not critical; the formation of product increased only slightly (less than 10%) when their concentrations were doubled.
Using the standard incubation mixture, the enzyme was less stable at 37" or 30" than at 25". Under standard conditions as well as with other glycerol concentrations in the assay mixture (compare Fig. 2) the enzyme activity was a linear function of time up to 10 min of incubation.
Formation of N-acetylglutamate depended in a linear manner on the amount of protein in the assay provided that not more than approximately 15% of [%]glutamate was converted to N-acetylglutamate.
Partial Purification of N-Acetylglutamate Synthetase by Xedimentation-When crude extracts were prepared with extraction buffer containing 10 mM magnesium acetate and centrifuged for 4 hours at 110,000 x g, approximately 50% of the enzyme activity was found in the pellet. Under these ionic conditions, the bulk of the enzyme sedimented at 40 to 50 S as determined by centrifugation through gradients of 25 to 40% (v/v) glycerol. If magnesium was omitted or Tris buffer was substituted for phosphate buffer, the enzyme could not be sedimented by centrifugation at 110,000 x g for 4 hours. Although it is not clear whether the enzyme undergoes a magnesium-induced association with itself or with some other macromolecules, the observed effect allowed the development of a procedure for a partial purification of the enzyme by centrifugation.
It consisted of two centrifugation steps and resulted in an approximately 6-fold purification of the enzyme with 40% recovery of the initial activity.
The first centrifugation was done in the presence of 10 mM magnesium acetate and led to a sediment with about 5Ooj, of the initial enzyme activity.
This pellet was dissolved in extraction buffer containing 0.1 M EDTA, incubated overnight at 4", and subjected to a second centrifugation at 110,000 X g for 4 hours. Over 807, of the activity recovered in the pellet of the first centrifugation were found in the supernatant of the second Z D. Haas and T. Leisinger, Eur. J. Biochem., in press. centrifugation. Inhibition of N-Acetylgluta.mate Synthase by Arginine and Arginine Analogues-A 6-fold purified preparation of the enzyme in extraction buffer containing 0.1 M EDTA was used for studying its inhibition by arginine, precursors of arginine, and various analogues. The substrate concentrations used in the inhibition experiments were saturating for crude extracts.
It was observed that inhibition by L-arginine and O-(L-norvalyl-5)-isourea reached a plateau value at about 15% residual activity, which could not be reduced further by increasing the inhibitor concentrations (Fig. 1). As explained earlier, the residual activity observed at high inhibitor concentrations was due to N-acetylglutamate synthase activity. In an effort to establish whether this residual activity represented a physiologically important property of the enzyme or whether it was due to the assay conditions, we tested the inhibition of N-acetylglutamate synthase by arginine at various concentrations of glycerol. Fig. 2 shows that the residual enzyme activity in the presence of 10 mM L-arginine was strongly dependent on the glycerol concentration in the incubation mixture. The enzyme activity in the absence of L-arginine, on the other hand, was only slightly depressed by raising the glycerol concentration in the assay. A similar modification of the feedback properties of the enzyme was observed  Because of its low level of argininosuccinase this strain converts L-ornithine at a growth restricting rate to L-arginine. The arginine restriction leads to full derepression of the arginine biosynthetic enzymes during the early phases of growth. At later stages in the development of the culture an increase in the growth rate and partial repression of the arginine enzymes were observed. Therefore, strain 961 was harvested when the culture had reached approximately 2 X lo8 cells per ml. The other strains were grown up to approximately 8 X 108 cells per ml. Crude extracts of the different cells were used for the determination of N-acetylglutamate synthase, N2-acetylornithine B-aminotransferase, and protein (see "Materials and Methods" as a function of the pH in the assay. With Tris buffer, pH 7.2, the residual activity in the presence of 1 mM L-arginine amounted to 4% whereas with Tris buffer, pH 9.0, it was 16% (data not shown).
Regulation of N-Acetylglutamate Synthase Formation by Arginine---In E. coli the formation of the arginine biosynthetic enzymes is repressed noncoordinately by the end product of the pathway. For seven out of the eight arginine biosynthetic enzymes the repression ratio has been established. It differs depending on the enzyme examined and is in the range between 3 and approximately 500 (22). Aq quantitative information about the control by L-arginine of N-acetylglutamate synthase is lack-ing, we determined the specific activity of this enzyme in relation to regulatory conditions. Table I lists the specific activities of  N-acetylglutamate synthase and, for comparison, of N2-acetylornithine 5-aminotransferase in the argR+ strain 619, grown on minimal medium with and without L-arginine.
These growth conditions lead to repression and to partial derepression, respectively of the arginine biosynthetic enzymes. Derepressed enzyme levels were obtained either genetically, by growing the argR-strain 619/14 on minimal medium with n-arginine, or physiologically, by growing the arginine auxotroph 961 on minimal medium with n-ornithine, which in this organism is converted at a growth restricting rate to n-arginine.
In repressed cells N-acetylglutamate synthase was not detectable (i.e. the specific activity was less than 0.2 unit per mg of protein). The derepressed enzyme levels amounted to approximately 50 units per mg of protein, which results in a repression ratio of greater than 250 (Table I). Strain 961 was grown in a Chemap bench fermenter wiih a 7-liter vessel, containing 6 liters of Medium E supplemented with the reauired amino acids plus 100 ug of L-ornithine-HCl per ml. Under-these conditions the arginme biosynthetic enzymes are fully or partially derepressed, depending on the stage of the culture (see Table I). When the culture had reached a densitv of approximately 5 X lo8 cells per ml, L-arginine-HCl was added to give a final concentration of 120 pg per ml (arrow).
At the times indicated, samples from the growing culture were rapidly cooled, mixed with a concentrated chloramphenicol solution to give a final concentration of 100 fig per ml, and used for the preparation of crude extracts. N-Acetylglutamate synthase and protein were determined in the crude extracts as described under "Materials and Methods." (O--O), specific activity of N-acetylglutamate synthase as determined experimentally; (O---0), specific activity of N-acetylglutamate synthase assuming dilution of pre-existing enzyme by growth (calculated). derepression (growth on L-ornithine) was followed by repression (addition of n-arginine). The specific activity of the enzyme was measured during early repression. Within the limits of the experimental error, the specific activity followed the pattern expected for the dilution of the enzyme by exponential growth. Thus, the absence of measurable enzyme activity in steady state repressed cells is probably due to the arrest of N-acetylglutamate synthase formation and not to inactivation of the enzyme.