REGULATION OF L-ISOLEUCINE BIOSYNTHESIS IN THE PHOTOSYNTHETIC BACTERIUM RHODOSPIRILLUM RUBRUM* BY CATHERINE NINGt AND HOWARD GESTt ADOLPHUS BUSCH III LABORATORY OF MOLECULAR BIOLOGY, WASHINGTON UNIVERSITY, ST. LOUIS, MISSOURI Communicated by Martin D. Kamen, October 17, 1966 Biosynthetic threonine deaminase (TD) catalyzes the conversion of L-threonine to a-ketobutyrate and is the first of a series of enzymes uniquely concerned with the synthesis of L-isoleucine in bacteria. Partial control of isoleucine production in such cells is provided through feedback inhibition of the deaminase by the end product, isoleucine.' The specific feedback properties of threonine deaminases, however, may differ significantly in different microorganisms. Thus, isoleucine is a potent inhibitor of the cell-free enzyme from Escherichia coli,2 Salmonella typhimu- rium,3 and Rhodopseudomonas capsulatus,4 even in the presence of relatively high threonine concentrations. Activity of the Rhodospirillum rubrum (strain Si) en- zyme, on the other hand, is appreciably inhibited by isoleucine only at low threonine concentrations, and the inhibition is gradually relieved as the substrate concentra- tion is increased.4 Although the in vitro behavior of the enzyme could be considered to reflect "loose" or inefficient feedback control of TD activity in R. rubrum,4 other interpretations are possible. This communication presents a re-evaluation of the feedback control pat- tern for synthesis of L-isoleucine and other amino acids of the aspartic family (threonine, methionine, and lysine) in this organism based, in part, on further study of the "native" TD and of an experimentally modified form with increased sensitiv- ity to feedback inhibition by isoleucine. Experimental.-The source of TD for the experiments reported here was a mutant strain of R. rubrum Si; the mutant, designated as S1H, was isolated from the cell population which even- tually developed in an anaerobic illuminated culture, in a medium containing 0.4% Lserine and 0.016% glycine as the only organic substrates. The mutant grows readily in malate plus am- monium salt media and shows a greatly elevated TD content as compared to the wild-type strain (approx. 15- to 20-fold). With glutamate as the nitrogen source, growth of the parent strain is markedly inhibited in the presence of exogenous L-threonine5 and as might be expected, growth of the mutant is much less susceptible to inhibition by this amino acid. Extracts were prepared from cells grown photosynthetically, under anaerobic conditions, in the synthetic malate + ammonium sulfate (1.25 gm/liter) medium described by Ormerod et al.' Since the TD of R. rubrum is unstable at 0-5°C under certain conditions,4 cells were harvested and all subsequent operations conducted at room temperature (unless otherwise noted). Follow- ing a single wash with 0.5 M potassium phosphate buffer pH 8.0 containing pyridoxal phosphate (25 ug/ml) and a low concentration of I-isoleucine (0.1 mM), the cells were disrupted by grinding with fine alumina according to the procedure of McIlwain.7 The ground mixture was extracted with 5 ml of the buffer for each gram of cell paste used. Alumina and cell debris were removed bylow-speed centrifugation, and the extract wasfurtherclarified bya second centrifugation at 65,000 X g for 4 hr (Spinco rotor no. 30). The extract was then heated at 600C for 10 min and coagulated proteinswereremoved bycentrifugation. Saturated ammonium sulfate solution, adjusted to pH 7.4 with concentrated ammonium hydroxide, was added dropwise to the heated fluid until 0.35 satura- tion was attained. The resulting precipitate was collected by centrifugation, triturated in the buffer noted to give a protein concentration of 5-10 mg/ml, and the preparation dialyzed for approximately 2 hr against a relatively large volume of buffer. At this stage, the enzyme was reasonably stable to storage at -15°C, about 50% of the activity remaining after 1 week. Ordi- 1823 Downloaded by guest on September 30, 2021 1824 BIOCHEMISTRY: NING AND GEST PROC. N. A. S. narily, the extent of purification was 30- to 40-fold. Papain-treated TD for the experiments of Figures 1 and 3 was prepared as follows: The deaminase, partially purified as described, was incubated with crystalline papain in a malonic acid (pH 6.5) + ,3-mercaptoethanol buffer (see details in legends) for 5-10 min at room temperature. TD was then separated from the papain by filtration of the mixture through a small Sephadex G-75 column (equilibrated with 0.05 M potassium phosphate buffer pH 8.0); additional 0.05 M buffer was added to the column, as necessary, to displace the deaminase. For the kinetic experiment of Figure 2, the column separa- tion was not employed. Deaminase activity was estimated by a modification of the procedure of Friedemann and Haugen.8 The reaction mixtures contained the following components, in a final volume of 1 ml: potassium phosphate pH 8.0, 100 Mmoles; pyridoxal phosphate, 5 sg; L-threonine, enzyme, and other additions as indicated. Following incubation for 30 min at 370C, the reaction was termi- nated by adding 0.3 ml of a 2: 1 mixture (v/v) of 0.2% dinitrophenylhydrazine hydrochloride (in 2 N HCl) and 30% trichloroacetic acid. After additional incubation for 10 min, 1 ml of 2.5 N NaOH was added. Ten min were then allowed for color development before measuring absor- bancy, at 540 my, in a Zeiss PMQ II spectrophotometer (1-cm light path). Blank reaction mix- tures contained all relevant components, except substrate. Enzyme activity is expressed in terms of absorbancy; a value of 1.0 is equivalent to 0.25 Mumole of a-ketobutyrate. Results and Discussion.-Feedback inhibition of the "native" S1H threonine deami- nase by isoleucine: The effect of isoleucine on activity of the "native" R. rubrum S1H deaminase at various substrate concentrations is illustrated by data in Figure 1 (solid lines). With 1 mM isoleucine, significant inhibition occurs only at threonine concentrations less than 3 mM. At higher isoleucine concentrations (e.g., 10 mM), the same general relationships of the curves (4± isoleucine) are observed, as reported earlier for the enzyme of the parent S1 strain.4 The sigmoidal character of the sub- strate-dependence curve, indicating cooperative effects frequently seen with regula- tory enzymes, is considerably exaggerated in the presence of isoleucine. Effect of papain on the S1H threonine deaminase: Taketa and Pogell9 have demon- strated that the regulatory properties of mammalian fructose-1,6-diphosphatase can be markedly altered by treatment of the enzyme with papain. The proteolytic enzyme progressively inactivates the diphosphatase, but appears to have a preferen- tial effect on binding sites for an allosteric effector, adenosine 5'-monophosphate. By appropriate exposure to papain, active diphosphatase which is no longer in- hibited by the effector can be obtained. Similar treatment of the R. rubrum S1H threonine deaminase results in the opposite effect, i.e., increase in sensitivity to the effector, isoleucine. This is shown in Figure 2 (see also Fig. 1). As the deaminase becomes inactivated, there is a rapid gain in feedback sensitivity to isoleucine at high substrate concentration (10 mM threonine in Fig. 2). Inactiva- 1.2- PAPAIN TREATED FIG. 1.-Feedback inhibition of R. ru- 1.0 brum S1H threonine deaminase activity >_ _ by L-isoleucine (IL). For the "native" F 0.8 _ enzyme curves (solid lines), the reaction P / mixtures each contained 13 Aig of protein; LU OA + lmM IL --A threonine and isoleucine were added at the Z __ concentrations indicated. Papain-treated was from LUZ 0.4 / Z __theenzymesame(dashedammoniumlines)sulfatepreparedfraction, using 0.20.2 0.04 mg papain/mg bacterial protein (in _ 0.2 M malonic acid + 0.07 M fl-mercapto- o I1. ethanol buffer); each assay mixture con- 0 1.0 2.0 3.0 4.0 tained 46 ,ug of protein. L-THREONINE CONC. (mM) Downloaded by guest on September 30, 2021 VOL. 56, 1966 BIOCHEMISTRY: NING AND GEST 1825 FIG. 2.-Time course of papain inactiva- 9J tion and simultaneous change in feedback \ ACTIVITY sensitivity of R. rubrum SLH threonine 80 deaminase to L-isoleucine. The deaminase 70 D (same ammonium sulfate fraction as used for X \ was incubated with at C in 60 Fig. 1) papain 370 ac 0.15 M malonic acid + 0.1 M ft-mercapto- * ethanol buffer (bacterial protein, 2 mg/ml; 50XINHIBITION* papain, 0.14 mg/ml). At 1-min-intervals, a 40 -\0.1-ml sample was diluted 100-fold in 0.5 M >_ @ < >potassium phosphate buffer (pH 8.0), and the I- 30 activity of 0.5 ml of the dilution immediately U. 2o assayed using 10mM L-threonine as substrate in the absence and presence of L-isoleucine ,, lo (IL, 20 mM). In this experiment, 100% ac- 0 (MI,VME ,tivity corresponded to an absorbancy of 0.86 01 2 3 4 S 6 in the assay. TIME (MIN) tion of the TD beyond a given point can be avoided by removing the papain, which can be accomplished by filtration of the incubation mixture through Sephadex G-75 (the mol wt of papain is considerably smaller than that of the deaminase). This procedure was used for the experiment of Figure 1 (dashed lines), and for Figure 3, which compares the sensitivities of "native" and papain-treated enzymes in respect to feedback inhibition by various concentrations of isoleucine at a constant and high level of threonine (10 mM). These results indicate that papain can modify the S1H deaminase to a form which resembles the more feedback-sensitive enzymes observed in a number of other bacteria. The action of papain apparently does not cause gross changes in molecu- lar size of the enzyme, as evidenced by the fact that significant differences could not be detected in sucrose density gradient centrifugation patterns of the two "forms." Treatment of the R.