Cooperative feedback control of barley acetohydroxyacid synthetase by leucine, isoleucine, and valine

ARCHIVES

OF

BIOCHEMISTRY

Cooperative

AND

BIOPHYSICS

Feedback

Synthetase

146, 542-550 (1971)

Control

of Barley

by Leucine,

Isoleucine,

Acetohydroxyacid and

Valine

B. J. MIFLIN Department

of Plant

Science, University

of Newcastle

upon Tyne, NE1 7RU., England

Received May 27, 1971; accepted July 7, 1971 Acetohydroxyacid synthetase has been extracted and partially purified from barley. The enzyme produces acetolactate with pyruvate as substrate and acetolactate and acetohydroxybutyrate with pyruvate and a-oxobutyrate as substrates. Negligible amounts of acetoin are produced. The rate of acetolactate production shows a sigmoidal relationship with substrate at low pyruvate concentrations, The enzyme is inhibited by leucine and valine singly, and cooperatively by the pairs leutine plus valine. Cooperative inhibition is defined as occurring when the total concentration of a combination of inhibitors required to cause 50y0 inhibition is less than that for the most effective single inhibitor. Certain other pairs of amino acids also appear to give a degree of cooperative inhibition. The results suggest that the enzyme has two inhibitor sites, one for leucine and one for valine. Maximum efficiency of inhibition is achieved when both sites are Wed. Reagents which react with sulphydryl groups inhibit enzyme activity at millimolar concentrations. Iodosobenzoate, p-chloromercuribenzoate, and mercuric chloride desensitize the enzyme to leucine and valine inhibition. The enzyme is also desensitized by assaying at high pH values.

In microorganisms and in higher plants leucine, valine and isoleucine are synthesized by a pathway involving a series of common enzymes which metabolize either the precursors of isoleucine or of valine. The first enzyme in this pathway, the acetohydroxyacid synthetase, can either catalyze the formation of acetohydroxybutyrate from pyruvate and Lu-oxobutyrate or acetolactate from pyruvate. The enzyme is normally assayed by the production of acet’olact’ate and is thus often referred to as acetolactate synthetase. In microorganisms and in plants the enzyme requires Mn2+ or Mg2+ and DPT as cofactors (1, 2) and in microorganisms it also requires FAD (3). The synthesis of branched-chain amino acids is regulated, in part, by control of this enzyme. In bacteria t’he enzyme is subject to multivalent repression by isoleucine, leucine, and valine (4). In certain bacteria and yeast t,he enzyme is also subject to endproduct inhibition by valine (5, 6). In higher

plants end-product inhibition by valine has been reported for peas (7) and by valine and leucine (2) in barley. In t’he latter case synergistic inhibition between the pairs of amino acids leucine plus valine and leucine plus isoleucine has also been demonstrated. This paper is a report of more detailed investigations into the nature of t,he enzyme system and its control in barley. MATERIALS

AND METHODS

Shoots of 5- to 7-day-old, dark-grown barley seedings were ground in an ice-cold pestle and mortar in 0.05 M phosphate buffer, pH 7.5, containing EDTA 5 xn~, MgClz 5rn~, valine 1 mu, and leucine 1 mu. The homogenate was then filtered and centrifuged at 20,000 g for 7 min. The supernatant fraction was brought to 25Y0 saturation with respect to (NH& SOI and stood for 1 hr. After centrifugation the precipitate was discarded and saturated (NHd)z SOa added to the supernatant fraction to give 50y0 saturation. After standing for 1 hr the precipitate was collected by centrifugation and redissolved in 0.05 M

542

CONTROL

OF ACETOHYDROXYACID

phosphate buffer, pH 7.5, EDTA 1 mu, 0.1 M NaCl. The preparation was then passed over a Sephadex G-50 column using the same buffer as the eluant. Further purification, when carried out, was done by batch treatment with DEAE-cellulose. In this case the (NHd)z SO4 precipitate was redissolved in 0.05 M Tris-Cl buffer, pH 7.8, and passed over the column in the same buffer. The enzyme solution was then treated with an equal volume of DEAEcellulose slurry which had been preequilibrated in 0.05 M Tris-Cl buffer, pH 7.8. After standing for 10 min the slurry was centrifuged, and the pellet was washed with 0.01 M pyruvate in Tris-Cl buffer. The pellet was then resuspended in 0.3 M pyruvate in Tris-Cl buffer and, after standing for 10 min, recentrifuged, and the supernatant fraction retained. The majority of the work reported here was done using the ammonium sulfate and G-50 purificat#ion steps only. The extraction was carried out either in the absence of leucine and valine or the homogenate passed over Sephadex prior to assay so that an estimate could be made of the activity of the crude homogenate. From this the increase in specific activity was found to be 15- to 30-fold :at the ammonium sulfate precipitate stage and 30- to go-fold after DEAE-cellulose treatment. The enzyme was assayed basically as described previously (2). The reaction mixture consisted of Na pyruvate 0.02 M, unless otherwise stated, DPT 0.32 mM MnSOa, 0.5 mM, phosphate buffer, 0.02 M, pH 7.0, and 0.4 ml enzyme in a final volume of 1 ml. After incubation at 30’ for 20 min, the reaction was stopped by the addition of 0.3 ml 0.2 N ZnSOd and 0.2 ml 1 N NaOH. After centrifugation the supernatant fraction was assayed for acetolactate by decarboxylation under acid conditions and subsequent estimation of the acetoin formed by the method of Westerfeld (8). Checks of direct acetoin formation during the enzyme assay were made, but since this was less than 5y0 of the acetolactate formed, this control was routinely omitted. Although the routine assay of activity was by acet,olactate production, the ability of the enzyme to form acetohydroxybutyrate with pyruvate and a-oxobutyrate as substrates was verified by forming the pteridine derivatives of the decarboxylated products of the reaction following the method of Wagner et al. (9). The derivatives were then identified by their RF values in a range of solvents (9). The spot corresponding to the known RF of 6 methyl-7-ethyl-2-amino-4-hydroxy pteridine, the chief product of the reaction between aceto-ethylcarbinol (produced by decarboxylation of acetohydroxybutyrate) and 2-, 4-, 5-triamino-6hydroxy pyridine-di-hydrochloride appeared only when or-oxobutyrate was included as a substrate. The amino acids and their analogs used in this

SYNTHETASE

study were all of the L from Sigma (London) highest purity available tographically pure in a

543

form and were purchased Ltd. They were of the and found to be chromarange of solvents.

RESULTS

E$ect of Pyruvate Concentration Although studies (5, 7) have shown the reaction kinetics with respect to pyruvate concentration to be first order, these have chiefly been carried out at high pyruvat’e concentrations giving minimum rates in excess of 20% of V,, . Preliminary experiment’s with the barley enzyme showed a sigmoidal relationship between substrate and rate at low pyruvate concentrations (2). In this experiment the pyruvate concentration went up to 1.5 mu, at which level the rate started to level off, even t’hough experiments over the higher concentration range continued to show an increase in rat,e up to 100 mM. Further detailed experiments were carried out at bot’h high and low concentration ranges. At the high concentration range the curve of rate versus pyruvate concentration approximates to a rectangular hyperbola (Fig. 1) and results in a linear reciprocal Lineweaver-Burk plot giving an apparent K, of 14 mM in agreement with the previous study. The low range (O-5 mM pyruvate) plot (Fig. 2) gives a sigmoid curve flattening at 1.5-3.0 nq as found previously, and then showing a second upward curvilinear increase in rate above 3.0 mM. This experiment was repeated a number of times and in each case a similar double-sigmoid relationship was observed. The position and degree of the “plateau” varied slightly from preparation to preparation. Extrapolating the plateau an apparent’ V,,, can be obt,ained and used to construct8 a Hill plot for the lower sigmoid curve (Fig. 3). The plot gives a straight line with a slope of n = 2.2. Such kinetics, particularly the presence of a sigmoid relationship, are characteristic of allosterically regulat’ed enzymes. These kinetics are also to be expected in cases in which the enzyme reacts with the same molecule twice in different ways as happens here. The lower sigmoid curve may thus reflect t’he binding of pyruvate at one enzyme site to give hydroxyethyl-DPT and the sub-

544

MIFLIN

I 0

I 02 Pyruvate

I 04 con‘“.

I 06 CM)

I 08

I 10

FIG. 1 FIGS. 1 and 2. The effect of pyruvate concentration on the rate of acetolactate production. The rates were determined over a lo-min assay.

I 10 Pyruvate

I 2” concn.

I 30 (mtvl

)

t 40

is highest at low concentrations and decreases wit’h increase in concentration with an antagonistic relationship existing above 0.75 mM. The classical definibion of cooperative inhibition, generally accepted in the literature, is attributed to Stadtman (10) and is defined as occurring when an excess of any of the ultimat,e end products causes a partial inhibition of the first enzymic step, whereas the simultaneous excess of two or more of the end products results in a greater inhibition than the sum of the fracbional inhibit#ions caused by each independently. By this criterion the inhibitors leucine and valine fail to qualify as cooperative since they are only synergistic at levels below “excess.” However, t’his definition is inappropriate since the organism is not normally concerned with saturating levels of feedback inhibitors. Indeed, the occurrence of saturating levels in the tissue would mean that the feedback control mechanism had broken down and was not fulfilling its function. The organism is, in fact’, best served by a syst’em in which large changes in rate occur with small changes in concentration of effecters and thus it is the change in the situation around the midpoint of t’he reaction rate that is most important. The relative efficiencies of the various inhibitors may be more meaningfully expressed in terms of either the concentration of inhibitor required to give 50% inhibition (150%) or the concenOration of inhibit’or re-

I 50 ?O-

FIG. 2

sequent binding of pyruvate at a second site where it react’s with the DPT complex to give acetolactate. The slope of approximately 2 in the Hill plot is consistent with this. Inhibition

by Amino Acids

7 > $oo-

E > t z _J io-

The degree of inhibition of the reaction caused by various concentrations of leucine, valine, and leucine plus valine is given in Fig. 4. The inhibition due to the presence of leucine plus valine is obviously greater than that due to either amino acid singly. Various ways can be used to quantify the relative efficiencies of the amino acids singly and in combination. The level of synergism (Fig. 5)

I

i 01. 10 Log

i5 pyruvate

t

I

00 concn

05 (mM1

FIG. 3. Hill plot of rate vs pyruvate concentration at values up to 2.0 mu. The data are taken from Fig. 2 using an apparent V,, of 0.7 pmoles/ mg protein/hr for the lower sigmoid curve.

CONTROL

OF ACETOHYDROXYACID

545

SYNTHETASE

quired to give half the maximal inhibit#ion caused by t’hat inhibitor (Mo.~). The values of the former criterion are shown in Fig. 4 and t’hose of the latter in Figs. 6 and 7. Of the two, Ir,Og~is related t,o the maximal level of inhibition. caused by the inhibitor as well

FIG. 4 FIGS. 4 to 7. Inhibition of acetolactate synthetase by leucine and valine. Figure 4. The percentage inhibition caused by increasing total concentrations of amino acid present as either leucine, valine, or leucine plus valine (1:l). Fig. 5. Variation in the synergistic inhibition of leucine and valine with differing concentrations. The percentage synergism is calculated au percentage of inhibition of leucine and valine together - (percentage of inhibition by leucine + the percentage of inhibition by valine), and the amino acid concentration of each individual amino acid is given. Figs. 6 and 7. Effect of various concentrations of leucine, valine, leucine plus valine (l:l), and valine in the presence of 0.25 mM leucine on the inhibition of acetolactate synthetase expressed as a percentage of the maximum inhibition obtained by each combination.

Concn

Of “allme

CrnM,

FIG. 7

as the slope of the inhibition curve, whereas M0.6 probably, but not necessarily, reflect’s the affinity of t,he modifier for the enzyme. However, whichever criterion is used, the pair leucine plus valine appear as much more effective inhibitors than either leucine or valine alone in t,hat they cause 50% inhibition of activity at a concentration approximately one order of magnitude less than t’hat of leucine which also has an Mo.s value six times great,er. In addition, the maximum level of inhibiCon is around 90% compared wit’h 80% due to leucine. The maximum levels of inhibition were obt’ained by extrapolating back to infinite inhibitor concentration reciprocal plots of percentages of inhibition versus inhibitor concentration. The interaction of leucine and valine can also be seen from the effect of the presence

MIFLIN

546

of one of t’hem on the inhibition caused by increasing concentrat’ions of the ot’her. Figure 7 shows the effect of the presence of leucine on the percentage of maximum inhibition due to valine caused by increasing concentrations of valine. Similar plots are obtained in the reverse situation of varying leucine concentrations in the presence and absence of valine. In both cases the presence of the second inhibitor decreases the MM values, thus leucine reduces the Mw, (val) from 0.45 mM to 0.04 rnM and valine the MO.5 (leu) from 0.275 rnM to 0.03 mM. The addition of leucine also changes the shape of the reciprocal plot of percentages of inhibition due to valine versus valine concentration. In the absence of leucine a nonlinear curve is obtained, whereas t,he addition of leucine results in a straight-line relationship of greatly decreased slope. The results obtained are generally indicat’ive of a first-order reaction with respect to valine in the presence of leucine. A similar conclusion can be drawn for leucine in the presence of valine. Although leucine and valine cooperate in inhibiting the enzyme, there is little evidence of binding interaction in the percentage inhibition versus concentration curves (Fig. 4). Only in the case of the single inhibitors (e.g., leucine) is a small sigmoidal effect observed at low concentrations. This lack of interaction is confirmed from Hill plots of these data (Fig. 8) which, although curved, give midpoint values near to n. = 1, only at low values of leucine does the slope become

2. Further lack of interaction was obtained by treating the data as suggested by Atkinson’s modification (11) of plots originally utilized by Tagata and Pogel (12), again values close to 1 are obtained. The 150%and M0.6 values given above are not absolute constants but are dependent on the substrate concentration. This is shown by t,he results of a series of experiments on the effect of pyruvate concentration on the Mo.s values and level of maximum inhibition caused by leucine plus valine (Table I). Although the major effect of increasing the pyruvate concentration is to increase the Mo,r, there is also a decrease in the level of maximum inhibition. Similarly, although it has been shown previously (2) that the effect of leucine and valine is largely competitive in that the major effect of leucine and valine is on the apparent K, for pyruvate, there is also a small change in Vi,,, which increases with increasing leucine plus valine concentrations. The system, therefore, approximates to, but does not completely fit a ‘K system’ as defined by Monad et al. (13). Specificity

of Inhibition

Various other amino acids have been tested for t’heir effect on acetolactate synthetase and the results are shown in Table II. Any inhibitory effects are largely confined to analogs of leucine and valine and occur only at relatively high concentrations. TABLE

I

THE EFFECT 0F PYRUVATE

CONCENTRATION ON THE LEVEL OF MAXIMUM INHIBITION AND THE CONCENTRATION OF LEUCINE PLUS VALINE REQUIRED TO GIVE HALF-MAXISWM INHIBITION” Pyruvate

conce(nt;ation Expt. number Y

Log

anhlbttor

concn

(mM

1

FIQ. 8. Hill plot of rate vs inhibitor concentration, where 211is the rate in the presence and Q the rate in the absence of the inhibitor.

0.01 0.02 0.05 0.1 0.1 0.2

1 2 3 1 3 2

Approx. max.

MO.6for leu(~+&

95 90 90 90 85 80

0.03 0.04 0.03 0.06 0.06 0.10

inhibition (%)

a The table is derived from three separate using experiments different preparations of enzymes.

CONTROL

OF ACETOHYDROXYACID TABLE

INHIBITION

Amino acid

Leucine Leucine Valine Valine Nor-valine Isoleucine (YAmino-n-butyrate Nor-leucine (YAmino-iso-butyrate Alanine Homoserine Aspartate Methionine

SYNTHETASE

547

II

OF ACETOLACTATE SYNTHETASE BY VARIOUS AMINO ACIDS SINGLY AND IN COMBINATION Concentration (mM)

Percentage inhibition Alone

0.1 0.5 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 10.0 10.0 10.0

26 41 22 30 20 16 15 14 4 2 11 11 15

+ Val (0.1 m&I)

f Leu (0.1mu)

9oa

50 32 22 6.2 28 15

85 41 68 56 37 27 35

0 Those combinations showing a marked degree of synergism are in italics.

The abilit)y of the various analogs to COoperate with either leucine or valine was measured by testing for a synergistic relationship in the presence of 0.5 mM of the amino acid plus 0.1 mu leucine or valine. In general, amino acids tend to cooperate with either leucine or vaIine, but not both. On the basis of the results in Table II and similar experiments it was found that nor-valine, nor-leucine, and leucine cooperate with valine, while isoleucine, valine, and cu-aminon-butyrate cooperate with leucine. The results suggest that there are at least, two inhibitor binding sites on the enzyme with different specifi ties. The Effect oj pH on Reaction Rate and Degree of Inhibition Bacteria possess two enzyme systems capacetolactate from able of producing pyruvate. These two systems have different pH optima and serve different functions. The pH 8 enzyme appears to be concerned with amino acid biosynthesis and is subject to feedback inhibition by valine or to multivalent feedback repression by leucine, isoleucine, valine (1). The pH 6 enzyme is not apparently involved in amino acid biosynt,hesis, it produces acetoin directly and is considered as part of a pyruvate oxidase complex (14). Davies (7), working with a pea-seed enzyme, suggested that there may

/A!10 d I FIG. 9. Effect of pH on the rate of acetolactate synthesis at A-A, 0.1 M, and O-O, 0.005 M pyruvate concentrations.

also be two acetolactate synthetases in higher plants. This deduction was based chiefly on the observation that his system had two pH optima, the relative importance of which differed with pyruvate concentration. At pH 8-8.5 the system showed a much higher affinity for pyruvate than at pH 6 to 7. The barley enzyme also shows two pH optima, alt’hough this does not differ wit’h pyruvate concentration (Fig. 9). There is no change with pH in the proportion of acet.oin

548

MIFLIN

formed directly in bhe reaction. Further studies showed that, consistent with this, the apparent K,,, for pyruvate changes little with pH. The degree of inhibition caused by amino acids, however, is affected by pH (Table III). In all cases there is a greater degree of inhibition at lower pH. The relative amounts of inhibition by valine, leucine, and valine plus leucine, is not affected. The system, therefore, differs from the bacterial one in having the greatest degree of feedback inhibition at lower pH. E$ect of Sulfhydryl

inhibited activity to some degree at millimolar concentrations. The major difference is that with some compounds, e.g., pCMB, HgClz , and iodosobenzoate, this inhibition is accompanied by a decrease in the level of inhibition due to leucine plus valine, whereas in ot)hers the level of inhibition stays the same. The former reagents, in general, result in the destruction of -SH groups either by oxidation or mercaptide formation, whereas the latter, by reduction lead to the formation of -SH groups. The degreeof desensitization to feedback inhibit)ion caused by HgCls increases with increasing pyruvate concentration (Table V). At high concentrations of pyruvate and HgClz , leucine and valine have no inhibitory effect in the presence of HgClz and may even cause a slight stimulation of rate.

Reagents

Acetolactate synthetase is responsive to a variety of reagents reacting with sulfhydryl groups (Table IV). All t’he reagents tried TABLE

III

DISCUSSION

THE EFFECT OF ASSAY pH ON THE PERCENTAGE INHIBITION BY LEUCINE AND VALINE Amino acid

PH

b&&&a)-

Leucine Valine Leucine + valine (1: 1)

1.0 1.0 0.5

Acetohydroxyacid synthetase is the first enzyme unique to leucine, isoleucine, and valine biosynthesis because it catalyzes the formation of both acetohydroxybutyrate and acetolactate. This means that for regulatory purposes the pathway can be considered as branched. The common first enzyme is then an obvious point for multivalent control. A number of ways have evolved of regulating the flow of carbon into branched pathways in such a manner that no one-end metabolite becomes limiting. In

6.6

7.0

7.9

8.4

715 66 83

51 44 76

40 30 66

38 21 57

0 Percentage inhibition of acetolactate synthetase. Assay solution contained 0.04 M phosphate and 0.04 M Tris, and the pH was adjusted to the above values with NaOH or HCl. TABLE

IV

THE EFFECTS OF SULFHYDRYL REAGENTS ON THE ACTIVITY AND FEEDBACK INHIBITION OF ACETOLACTATE SYNTHETASE Concentration of compound(mx)

Compound 0

pCMB control + Leu + Val HgClz + Leu + Val Iodosobenzoate + Leu + Val Mercaptoethanol + Leu + Val Glutathione + Leu + Val

lOOa 54b 100 56 100 53 100 48 100 54

0.01

0.05

100 59

78 43

116 54 102 54

98 49 109 54 97 55

0.1

0.05

1.0

74 39 69 32 98 47 78 45 96 56

59 30

10 0 43 -8 73 39 43 65 38 64

85 40 52 42 59 49

2.0

40 -4 69 37

0 Relative rates of acetolactate production in the presence of various concentrations of the reagents. b Percentage inhibition caused by leucine plus valine (0.1 mM) in the presence of various concentrations of the reagents. Pyruvate concentration 0.02 M.

CONTROL TABLE THE

OF ACETOHYDROXYACID

V

EFFECT OF PYRUVATE CONCENTRATION THE D:ESENSITIZATION OF FEEDBACK INHIBITION OF ACETOLACTATE SYNTHETASE Pyruvate

concentration

ON

(x)

Treatment

Control +HgClz (0.5 mM) +Leu + Val (0.5 rnM) +HgCls + Leu + Val 0 Rate in micromoles gram protein/hour.

0.01

0.02 __~__

0.05

0.1

1.45” 0.30 0.30

1.88 0.66 0.37

2.02 1.38 0.65

2.10 1.40 0.99

0.18

0.29

1.13

1.62

acetolactate

formed/milli-

bacteria t,his pathway is chiefly controlled by concerted multivalent repression of acetohydroxyacid synthetase and some of the succeeding enzymes in the pathway by leucine, valine, and isoleucine. In some organisms there is also feedback inhibition of the enzyme by valine. In barley enzyme repression does not occur (Miflin, unpublished) but, as described here for barley and elsewhere (15) for a range of higher plants, acetohydroxy acid synthetase is subject to cooperat,ive feedback control by leucine plus valine, and leucine plus isoleucine. Flow of carbon t#o isoleucine is also controlled by feedback on threonine deaminase (16, 17) so that the evolution of leucine and valine as the most effective pair of regulators for acetohydroxyacid synbhetase ensures that the pathway is completely shut off in the presence of an excess of all three amino acids. In the presence of an excess of isoleucine only, t)he synthesis of leucine and valine can continue unaffected. Although the presence of a large excess of exogenous leucine can inhibit the enzyme and lead bo inhibit’ion of growth (18), this is unlikely to occur in vivo as the rate of leucine biosynthesis is also regulated by feedback inhibition of isopropyl-malate synthetase (19). The nat,ure of the inhibition by leucine and valine singly and together is not one of unambiguous synergism but rather it is that together they are effectjive at a much lower concent,ration than either singly. This can

SYNTHETASE

549

be explained by one of at least, two possibilities. Firstly the evidence of the inhibition curves for leucine plus valine and valine in the presence of leucine (and vice versa) does not suggest that more t,han one site is present’ for each of the two inhibitors. The analog studies, and the fact that any given analog can cooperate with either leucine or valine, but not both, also suggests that there is one inhibitor site for leucine and its analog and one for valine and its analog. One possibility is that) the enzyme is inhibited only when both the inhibitory sites on the enzyme are filled (Fig. 10, Scheme Ia). Because of the structural similarity between leucine and valine, it is possible that, besides binding to their own site, they also have a low affinity for the ot’her’s site. Thus, at high concentrations of leucine (or valine) both sites become filled with leucine (Fig. 10, Ib) and inhibition occurs. An alt)ernative possibility (Fig. 10, II) is that binding of one inhibitor produces only partial inhibition, full inhibition depending on the binding of both inhibitors. It is possible that the presence of one inhibitor may increase the enzyme’s affinity for the second. An approximation of the affinity of the enzyme for the inhibitors is probably related to the MO.5 values and t’hese have been used in the schemes. In classic terms the first hypothesis is in accordance with the mechanisms outlined by Monad et al. (13), whereas the second is more in accordance with the theories of Koshland et al. (20). However, no SCHEME I

FIG. 10. Possible models for the mechanism of cooperative inhibition of acetolactate synthetase. k is the concentration at which, given a constant substrate concentration, the inhibitor site is halfsaturated with amino acid. The values given are approximations based on Mo.s values obtained with 0.02 M pyruvate as sub&ate.

550

MIFLIN

real Darallel can be made in bhe absence of knowledge of the subunit structure and inhibitor-binding properties of the enzyme. The kinetic studies reported here suggest that the first scheme is more probable since it would predict that at high concentrations of leucine the level of inhibition would approach the maximum (approx. 90%) and that,, whereas there might be some evidence of sigmoidicity in the inhibition curve of leucine alone, none would be expected in the presence of excess valine. In common wit’h most regulatory enzymes acetohydroxyacid synthetase can be desensitized by treatment with sulfhydryl reagents. Inhibit,ion of enzyme act.ivity by reducing and alkylating reagents suggests that catalytic activity depends on the ability to form, or the presence of, S-S bonds. In contrast, the inhibitor site is unaffe&ed by reducing reagents but is desensitized by alkylating or oxidizing agents suggesting that -SH bonds are necessary for its activity. Similar results have been obtained with the Salmonella enzyme (1). Increasing pH also relieves inhibition without greatly affecting enzyme activity. It is interesting that feedback sensitivity in higher plants is greatest at the lower pH, whereas in Salmonella the feedback-sensitive enzyme has a high pH optimum and inhibition by valine falls with the pH (1). However, the Neurospora (21) and yeast (22) synthetases have pH optima of 7.0-7.5 similar to barley. The yeast enzyme is also desensitized by high pH as valine inhibition is maximal at pH 7 dropping to zero at pH 8.0, although activity is still around 50% of the optimum (6). An alternative conclusion for our results is that there are two enzymes with different pH optima, only one of which is subject to feedback, and they would then merely reflect the differing activities of t’he two enzymes. At the present time this is not considered likely.

ACKNOWLEDGMENTS The author gratefully acknowledges the financial support of the A.R.C. of Great Britain and the excellent technical assistance of Mrs. A. Wilkins. REFERENCES FHEUNDLICH, M., ST~RMER, 1. BAUERLE, R.H., F. C., END UMBARGER, H. E. Biochim. Biophys. Acta 92, 142 (1964). 8,227l (1969). 2. MIFLIN, B. J., Phytochemistry 3. ST~RMER, F. C., AND UMBARGER, H. E., Biochem. Biophys. Res. Commun. 17, 587 (1964). 4. FREUNDLICH, M., BURNS, R.O., ANDUMBARGER, H. E., Proc. Aat. Acad. Sci. U.S.A. 48, 1804 (1962). 5. LEAVITT, R. I., AND UMBBRGER, H. E., J. Bacterial. 83, 624 (1962). MAGEE, P.T., ANDDE-ROBICHON-SZULMAJSTER, H., J. Biochem. 3, 507 (1968). DAVIES, M. E., Plant Physiol. 39, 53 (1964). WESTERFELD, W. W., J. Biol. Chem. 161, 495 (1945). WAGNER, R. P., BERQUIST, A., AND FORREST, H. S., J. Biol. Chem. 234, 99 (1959). 10. STADTMAN, E. R. Advan. Enzymol. 28, 41 (1966). 11. ATKINSON, D. E. Annu. Rev. Biochem. 36, 88 (1966). 12. TAGATA, K., AND POGELL, B. M., J. Biol. Chem. 240, 651 (1965). 13. MONOD, J., WYMAN, J., AND CHANGEUX, J.P., J. Mol. Biol. 12,88 (1965). 14. HALPERN, Y. S., AND UMBARGER, H. E., J. Biol. Chem. 234, 3067 (1959). in 15. MIFLIN, B. J., AND CAVE, P. R., manuscript preparation. 9,959 (1970). 16. DOUGALL, D. K., Phytochemistry 17. SHARM~, R. K., AND MAZUNDER, R., J. Biol. Chem. 246, 3008 (1970). 18. MIFLIN, B. J., J. Exp. Bot. 20,810 (1969). Biophys. Acta 111, 79 19. OAKS, A., Biochim. (1965). 20. KOSHLAND, D.E., NEMETHY,G.,AND FILMER, D . , Biochemistry 6, 365 (1966). n, CAROLINE, D. F., HBRDING, R. W., KUWANA, LI.. H., SATYANARAYANA, T., AND WAGNER, R. P., Genetics 62, 487 (1969). 22. MAGEE, P. T.,ANDDE-ROBICHON-SZULMAJSTER, H., Eur. j. Biochem. 3, 502 (1968).