RHODOSPIRILLUM RUBRUM*, T Regulation of Biosyntheses Can Be

Home , Alcohol dehydrogenase, Homoserine dehydrogenase

VOL. 51, 1964 BIOCHEMISTRY: DATTA, GEST, AND SEGAL 125

Apgar, J., and R. W. Holley, Biochem. Biophys. Res. Comm., 8, 391 (1962). 12 Berg, P., F. H. Bergmann, E. J. Ofengand, and M. Dieckmann, J. Biol. Chem., 236, 1726 (1961). 13 Berg, P., and U. Lagerkvist, Acides Ribonucleiques et Polyphosphates (Paris: Centre National de la Recherche Scientifique, 1962). 14 Martin, R. G., J. H. Matthaei, 0. W. Jones, and M. W. Nirenberg, Biochem. Biophys. Res. Comm., 6, 410 (1962). 15 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 441 (1962). 16Weisblum, B., S. Benzer, and R. W. Holley, these PROCEEDINGS, 48, 1449 (1962). 17 von Ehrenstein, G., and D. Dais, these PROCEEDINGS, 50, 81 (1963). 18 Sueoka, N., and T. Yamane, these PROCEEDINGS, 48, 1454 (1962). 19 Warner, R. C., and P. Vaimberg, Fed. Proc., 17, 331 (1958). 20 Craig, L. C., and D. Craig, "Physical methods of organic chemistry," in Technique of Organic Chemistry, ed. A. Weissberger (New York: Interscience, 2nd ed., 1956), vol. 3, p. 149. 21 Bennett, T. P., J. Goldstein, and F. Lipmann, these PROCEEDINGS, 49, 850 (1963). 22 Bennett, T. P., J. Goldstein, and F. Lipmann, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), in press.

EFFECTS OF FEEDBACK MODIFIERS ON THE STATE OF AGGREGATION OF HOMOSERINE DEHYDROGENASE OF RHODOSPIRILLUM RUBRUM*, t BY PRASANTA DATTA, HOWARD GEST, AND HAROLD L. SEGALT HENRY SHAW SCHOOL OF BOTANY AND ADOLPHUS BUSCH III LABORATORY OF MOLECULAR BIOLOGY, WASHINGTON UNIVERSITY, AND DEPARTMENT OF PHARMACOLOGY, ST. LOUIS UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI Communicated by F. Went, October 11, 1963 Regulation of biosyntheses can be effected, in part, through control of enzyme activity by the phenomenon of feedback inhibition.1 A previous report2 from one of our laboratories established that the homoserine dehydrogenase of the photo- synthetic bacterium Rhodospirillum rubrum is subject to this type of control, and that inhibition of enzyme activity by threonine can be specifically reversed by metabolites which are biosynthetically derived from homoserine, namely, isoleucine and methionine. The present communication summarizes experiments which provide further insight into the molecular basis of the foregoing kinetic effects. Two independent approaches indicate that the feedback modifier3 threonine causes aggregation of homoserine dehydrogenase to a catalytically inactive form and that such aggregation is reversed by homoserine, isoleucine, or methionine. Materials and Methods.-Growth of bacteria: R. rubrum (strain S1) was grown photosyntheti- cally in the synthetic medium described by Ormerod et al.,4 but with 0.1% L-glutamate as the nitrogen source instead of ammonium sulfate. Enzyme preparation: Cell-free extracts were prepared by sonic disruption (10-kc oscillator), and the homoserine dehydrogenase was purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration; the detailed procedure will be published elsewhere. The preparations used had specific activities in the range of 6-10 units (see below) per mg of protein, representing purifications of the order of 200- to 300-fold with respect to crude extract. Enzyme assays: One unit of homoserine dehydrogenase is defined as the amount of enzyme catalyzing the oxidation of 1 ,umole of NADPH/min at 370 in an incubation mixture (final volume, Downloaded by guest on September 24, 2021 126 BIOCHEMISTRY: DATTA, GEST, AND SEGAL PROC. N. A. S.

3 ml) of the following composition: enzyme; potassium phosphate buffer, pH 6.8, 300 jumoles; NADPH, 0.33 mg; ASA, ca. 2 ,umoles; EDTA, 3 jumoles. For the density gradient centrifugation experiments, homoserine dehydrogenase activity was estimated by measuring the decrease in absorbancy at 340 mp in a Cary Model 11 spectrophotometer under the conditions noted above. In the Sephadex chromatography experiments, activity was assayed by following reduction of NADP in the presence of homoserine; increase in absorbancy (340 mu) was followed at 250 in a Zeiss PMQ II spectrophotometer using l-ml incubation mixtures containing: enzyme; Tris buffer, pH 8.4, 100 ,umoles; NADP, 0.3 mg; homoserine, 10 Mmoles; EDTA, 1 jomole. One unit of enzyme as defined above catalyzes the reduction of 0.02 pmole of NADP/min in the latter assay. The final concentrations of the amino acid modifiers in the assay solutions were always below the level where significant kinetic effects are observed. Alcohol dehydrogenase activity was assayed by measuring reduction of NAD in the Cary (340 mpu; 370) in 3-ml incubation mixtures containing: enzyme fraction; Tris buffer, pH 8.5, 30d' ymoles; NAD, 0.7 mg; 25% (v/v) ethanol, 0.1 ml; reduced glutathione, 3 jomoles. Density gradient centrifugations: 0.2 ml of solution containing approximately one unit of homoserine dehydrogenase was layered over 4.6 ml of a linear sucrose gradient (5-20% (w/v) in 0.01 M potassium phosphate, pH 7.2). Crystalline yeast alcohol dehydrogenase (16 j&g; Sigma Chemical Co.) was always included in the overlaying solution to provide an internal standard. When substrate and/or modifiers were present in the sucrose gradient, these were also added at the same concentrations to the enzyme solution. The tubes were centrifuged in the SW-39 rotor of the Spinco Model L for 16 hr in the cold at a velocity of 32,000-32,700 rpm. Each tube was then punctured at the bottom, and 24 or 25 twelve-drop fractions were collected for assay of homoserine and alcohol dehydrogenase activities. Typical results are presented in Figure 1.

0.04 A C 0.15

0.03 ~~~~~~~~~~~~~~~~~~~~~~~~~~~LI

00.02 0

zX ~~~~~~~~~~~~~~~~~~~~~~~~~0.05~~~~~~~~~~~~~~~0 LuJ 0 0 U :9 0.01

th- prsneadasneo5.enieh envle r ie byurv 0.1 C.Ezm0.2 0.3 ciiisae04 0.5 0.6 xrse0.7 0.8sasrac FRACTIONAL DISTANCE SEDIMENTED (FROM TOP) FIG. 1-Density gradient sedimentation of homoserine and alcohol dehydrogenases in the presence and absence threo1tof ne. Curve A, homoserine dehydrogenase in the absence of threonine. Curve B, homoserine dehydrogenase in the presence of 0.001 M L-threonine. The sedimentation diagrams of alcohol dehydrogenase, used as an internal standard, were virtually identical in the presence and absence of threonine; the mean values are given by curve C. Enzyme activities are expressed as absorbancy changes (at 340 mg) per minute per 0.03-mi aliquot of fraction. For experimental details, see Materials and Methods. Sephadext chromatography: Sephadex G-200 (bead form; Pharmacia Fine Chemicals) was fractionated to remove fines by backwash with water in a standard 1-liter separatory funnel, and particles retained at a flow rate of approximately 140 ml/min were used to pack the columns. Before packing, the gel was suspended in buffer (0.05 M potassium phosphate + 0.001 M EDTA, Downloaded by guest on September 24, 2021 VOL. 51, 1964 BIOCHEMISTRY: DATTA, GEST, AND SEGAL 127

adjusted to pH 6.8), allowed to settle, and the supernatant fluid decanted. This washing procedure was repeated several times, and the final suspension was permitted to pack under gravity flow; 44 X 1.4-cm columns were used. Prior to use, the column was equilibrated with buffer which also contained substrate and/or modifiers where indicated. 2-3 ml of enzyme preparation in the same buffer were added to the gel column and rinsed into the gel with two successive 0.3-ml portions of the buffer. The column was then eluted with corresponding buffer at a flow rate of about 3 ml/hr; 1.4-1.6-ml fractions were collected and assayed for homoserine dehydrogenase activity. All of the gel filtration experiments were performed at 4°. Results.-Effects of substrate and modifiers on sedimentation of homoserine dehydrogenase: These effects on the sedimentation rate of R. rubrum homoserine dehydrogenase in a sucrose density gradient are shown in Table 1.

TABLE 1 SEDIMENTATION OF R. rubrum HOMOSERINE DEHYDROGENASE IN A SUCROSE DENSITY GRADIENT: EFFECTS OF HOMOSERINE AND MODIFIERS Rate of Sedimentation Relative to That of Yeast Alcohol Dehydrogenase: Additional supplement Enzyme Enzyme + 0.001 M L-threonine None 0.83, 0.72, 0.76 1.25, 1.27, 1.19, 1.26, 1.17 L-homoserine (0.02 M) 0.76, 0.78 ... 0.84, 0.87 ......

L-isoleucine (0.01 M) 1.00, 1.11, 1.02 1.08, 1.14 ...... L-methionine (0.01 M) 1.00, 1.04 ... 1.09, 1.09 ......

Under the conditions used, the rate of sedimentation of the homoserine dehydrogenase in the absence of substrate or modifiers is approximately 0.8 that of yeast alcohol dehydrogenase. In the presence of threonine, the sedimentation rate is markedly increased, to about 1.25. Calculations5 based on these relative sedimentation rates and the results of the gel filtration experiments described below suggest that the uninhibited enzyme exists as a monomer,6 and that the feedback inhibitor causes aggregation to a dimeric form. The amino acid modifiers, isoleucine and methionine, increase the sedimentation rate of the homoserine dehydrogenase significantly, but not to the extent observed with threonine. In this connection it is relevant that both isoleucine and methionine are activators of the enzyme in both crude extracts and purified preparations.' The effects of these amino acids on the sedimentation rate may reflect conformational alterations, other than aggregation, resulting in changes of shape or hydration of the enzyme,8 or an intermediate state of aggregation.9 Inhibition of the homoserine dehydrogenase activity by threonine follows apparent competitively kinetics with respect to homoserine and, as expected, the effect of threonine on sedimentation of the enzyme is completely reversed when sufficient homoserine is present. Aggregation due to threonine also appears to be prevented by isoleucine or methionine (both of which also reverse the kinetic effect of thre- onine2), since with these supplements the sedimentation rates are virtually the same in the presence and absence of the feedback inhibitor. L-lysine, on the other hand, does not influence enzyme activity and also has no effect on the density gradient sedimentation pattern in the absence or presence of threonine. Effects of substrate and modifiers on the molecular size of homoserine dehydrogenase as revealed by Sephadex chromatography: The results of the density gradient centrifugation experiments indicate that in the presence of the feedback inhibitor there is an increase in molecular size of the enzyme. Accordingly, the monomeric and aggregated forms should, in principle, be distinguishable by chromatography on appropriate Sephadex gels. Downloaded by guest on September 24, 2021 128 BIOCHEMISTRY: DATTA, GEST, AND SEGAL PROC. N. A. S.

A E 2.4 -3.0- CONTROL THR 0 C 1.6 2.0 _- |T\ 0.8 1.0_

B F X 2.4 6.0 o wJ THR 0 HSi LU I.1EU ML EFFLUN 0 E5.0-L b ~~~~+THR ZO ..3 Z4.0- &ILEU 5-0 -3.0- C 2.4-n +THR & HS(0.01M) 2.0 ~~~10~~~~ 10 2 010 4 0 0 30 0 S0 5- 0-~~~~~~~~~~~~~~~~~ L08 'U G 0 40. Bad thu D.thecur.resere obtaine FrseparTHR +MEt H D ~~~~~~~~~~&METH S 2.4 -3.0- THR & HS(0 02M) 1.6 20

ZIE)0.0110 20 Me 30 I-ehinn40 50 (MT)010 m20 .1M30 40 -o50 ML EFFLUENT ML EFFLUENT FIG. 2.-Elution diagrams of homoserine dehydrogenase from Sephadex G-200 in the presence and absence of substrate and/or modifiers. "Control" refers to enzyme in buffer alone. Where indicated, modifiers and substrate were present at the following concentrations: L-threonine (THR), 0.001 M; L-isoleucine (ILEU), 0.01 M; L-methionine (METH), 0.01 M; L-homoserine (HS), 0.01 or0s02 M as shown. In B, and E through G, the curves were obtained from separate experiments and have been placed in juxtaposition to facilitate comparisons. In the absence of threonine, the homoserine dehydrogenase is not retained by Sephadex G-100. With G-200, however, the enzyme is retained and, as shown in Figure 2A, upon elution the peak of homoserine dehydrogenase is in the fraction corresponding to about 0.6 of the column volume. When threonine is present, on the other hand, the enzyme is eluted from a G-200 column within the exclusion volume (Fig. 2B). This striking difference is interpreted as further evidence for the conclusion that threonine effects a polymerization of homoserine dehydrogenase. It may be noted that with suitable Sephadex gels (viz., G-150 and G-200), Peder- sen'2 has separated monomers of bovine serum albumin from its various poly- meric forms. -Isoleucine and methionine individually cause slight, but significant, displacement of the filtration diagram on Sephadex G-200 (Fig. 2F and 2G). This effect supports the conclusion drawn from the density gradient centrifugation expereiments that these amino acids induce changes in the physical state of the enzyme. If the inhibition of homoserine dehydrogenase activity by threonine is- attrib- utable to conversion of an active monomer to an inactive dimer, agents which reverse the kinetic effect should reverse the aggregation, and this should be detectable by gel chromatography. Experiments showing the effects of homoserine, isoleucine, and methionine on the molecular size distribution of homoserine dehydrogenase t~reated with threonine are summarized in Figure 2. From Figure 2C and 2D it is evident that with homoserine and threonine, two distinct ppaks are observed in Downloaded by guest on September 24, 2021 VOL. 51, 1964 BIOCHEMISTRY: DATTA, GEST, AND SEGAL 129

positions closely corresponding with those of the monomeric and aggregated forms of the enzyme (cf. Fig. 2A and 2B). With isoleucine and threonine (Fig. 2F), the high molecular weight peak characteristic of the threonine-treated enzyme is still present, but there is a considerable tailing which extends over the region where the lower molecular weight species would be expected; virtually identical results are obtained when the developing buffer contains both methionine and threonine (Fig. 2G). Although much higher concentrations of enzyme and lower pH values were used in the gel filtration and density gradient centrifugation studies than in the kinetic experiments reported earlier,2 it was found that these alterations in the experimental conditions did not significantly change the effects of the amino acid modifiers on the activity of the enzyme.7 Furthermore, other experiments indicated that under the assay conditions used (i.e., with threonine diluted to low concentrations), previously aggregated enzyme showed full activity and was essentially unaltered with respect to the kinetic effects of amino acid modifiers. Discussion.-Control of the rate of an enzymatic reaction by substances with structures substantially different from the normal substrates has been attributed to allosteric'3 modification of the enzyme molecule. It has been postulated13 that such modifiers bind at sites distinct from the substrate-binding sites, producing conformational alterations in the enzyme which, in turn, result in configurational changes in the active site. The results described in this communication indicate that feedback modifiers of the homoserine dehydrogenase of R. rubrum markedly affect the physical state of the enzyme. Several feasible mechanisms can be visualized for the apparent aggregation effected by threonine.'4 This amino acid may cause conformational alterations which lead to polymerization, but it is also possible that the threonine-dependent aggregation occurs through a process which does not involve changes in conformation of the monomeric units. It remains to be determined whether the binding, per se, of threonine to the enzyme causes loss of activity Qr whether aggregation is required for inhibition. The results of the density gradient centrifugation and gel filtration experiments are consistent with the conclusion that isoleucine and methionine also affect the physical state of the R. rubrum enzyme. These amino acids not only reverse the kinetic and aggregational effects of threonine, but also stimulate enzyme activity in the absence of threonine.2 7 The effects of isoleucine and methionine could reflect an independent structural modification or a competition for the threonine- binding site. In either event, it is evident that the presence of isoleucine or methionine leads to a form of the enzyme in which the active site is not unfavorably altered and which is not aggregated to the extent observed with threonine alone. Martin'5 and Utter'6 have looked for possible effects of feedback modifiers on the sedimentation characteristics of phosphoribosyl-ATP pyrophosphorylase and pyruvic carboxylase, respectively, but with negative results. Although the "de- sensitized" forms of Escherichia coli aspartic transcarbamylasel7 and homoserine dehydrogenasel8 exhibit decreased sedimentation rates as compared with the "native" enzymes, these changes have not been related to the kinetic effects of allosteric modifiers. The present studies provide evidence for a direct role of reversible association-dissociation phenomena in the regulatory action of amino acid modifiers on the homoserine dehydrogenase of R. rubrum, and it may well be Downloaded by guest on September 24, 2021 130 BIOCHEMISTRY: DATTA, GEST, AND SEGAL PROC. N. A. S.

that additional examples of feedback control through monomer-polymer interconversions will be found in other systems.'9 Summary.-The mechanism of control of Rhodospirillum rubrum homoserine dehydrogenase activity by amino acids derived from homoserine has been studied using techniques of density gradient centrifugation and gel filtration. The centrifugation experiments indicate that threonine causes aggregation of the enzyme to a catalytically inactive form, presumably a dimer, characterized by markedly increased sedimentation rate. Aggregation of the dehydrogenase in the presence of threonine also results in extensive displacement of the filtration profile on Sephadex G-200, permitting distinction of the two forms of the enzyme. The L-isomers of homoserine. isoleucine, and methionine reverse the feedback inhibition of catalytic activity by L-threonine and also reverse the threonine-induced aggregation. These observations are interpreted as evidence that monomer-polymer interconversions play a decisive role in regulating the activity of the homoserine dehydrogenase. The authors are grateful to Drs. Simon Black and Charles Gilvarg for gifts of aspartic a-semialdehyde. They are also indebted to Emmapaola Sturani, who participated in the early phase of this investigation, and to Joyce Henry and Robin A. Segal for expert technical assistance. * Supported by grants from the National Science Foundation (G-9877) and the National Institutes of Health (E-2640 and A-3642). t Abbreviations are: ASA, L-aspartic ,-semialdehyde; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP; EDTA, ethylenediaminetetraacetic acid, sodium salt; Tris, tris(hydroxymethyl)aminomethane. I Career Development Awardee (GM-5111) of the National Institutes of Health. 1 Moyed, H. S., and H. E. Umbarger, Physiol. Rev., 42, 444 (1962). 2 Sturani, E., P. Datta, M. Hughes, and H. Gest, Science, 141, 1053 (1963). 3 Feedback inhibitors or metabolically related substances which specifically modify enzyme activity, e.g., by inhibition, reversal of inhibition, or activation. 4Ormerod, J. G., K. S. Ormerod, and H. Gest, Arch. Biochem. Biophys., 94, 449 (1961). 5 Martin, R. G., and B. N. Ames, J. Biol. Chem., 236, 1372 (1961). 6 As used here, the term "monomer" refers to the form which exists in the absence of modifier without implication as to size or number of subunits. 7Datta, P., H. L. Segal, E. Sturani, and H. Gest, unpublished observations. 8 Schachman, H. K., Ultracentrifugation in Biochemistry (New York: Academic Press, 1959). 9 For example, if the monomer consists of two subunits, an intermediate aggregated state would be composed of three subunits. 10 When the reaction is measured in the other direction (ASA + NADPH - Homoserine + NADP), however, inhibition by threonine follows apparent noncompetitive kinetics with respect to ASA.7 Similar complex kinetic behavior has also been reported for yeast homoserine dehydrogenase. " "Karassevitch, Y., and H. de Robichon-Szulmajster, Biochim. Biophys. Acta, 73, 414 (1963). 12 Pedersen, K. O., Arch. Biochem. Biophys., Suppl. 1, 157, (1962). 13 Monod, J., J-P., Changeux, and F. Jacob, J. Mol. Biol., 6, 306 (1963). 14 Although it is conceivable that the described effects of modifiers could be due to unusual and extensive intramolecular configurational changes of the enzyme, other than aggregation, this possibility is considered to be unlikely. 16 Martin, R. G., J. Biol. Chem., 238, 257 (1963). 16 Utter, M. F., in Abstracts, 145th Meeting, American Chemical Society, September 1963, p. 55-c. 17 Gerhart, J. C., and A. B. Pardee, J. Biol. Chem., 237, 891 (1962). 18 Patte, J-C., G. LeBras, T. Loviny, and G. N. Cohen, Biochim. Biophys. Acta, 67, 16 (1963). 19 E. Scarano, G. Geraci, A. Polzella, and E. Campanile [J. Biol. Chem., 238, PC 1556 (1963) ] have recently suggested the possibility of a "molecular change" of 2'-deoxyribosyl 4-amino- pyrimidone-2,5'-phosphate aminohydrolase in the presence of modifiers. Downloaded by guest on September 24, 2021