Comparison between Thifensulfuron Methyl-Induced Inactivation of Barley Acetohydroxyacid Synthase andEscherichia coliAcetohydroxyacid Synthase Isozyme II

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO.

56, 231–242 (1996)

0076

Comparison between Thifensulfuron Methyl-Induced Inactivation of Barley Acetohydroxyacid Synthase and Escherichia coli Acetohydroxyacid Synthase Isozyme II FLORENCE ORTEGA,* JEAN BASTIDE,*,1

AND

TIM R. HAWKES†

*CNRS URA 461, Centre de Phytopharmacie, Universite´ de Perpignan, 52 avenue de Villeneuve, 66860 Perpignan Cedex, France; and †ZENECA Agrochemicals, Jealott’s Hill Research Station, Bracknell, Berkshire RG12 6EY, United Kingdom Received September 25, 1996; accepted December 23, 1996 Thifensulfuron methyl, a sulfonylurea herbicide, inhibited acetohydroxyacid synthase (AHAS) from Escherichia coli (isozyme II) and barley in a similar, time-dependent, manner. This was modeled in terms of a slow first-order transition (0.036 and 0.048 min01, respectively) from an initial, relatively weak complex of the enzyme with inhibitor to a final, more potently inhibited form. AHAS from both barley and E. coli appeared permanently inactivated. In neither case was activity recovered following removal of the inhibitor by gel filtration or dilution. However, removal of the inhibitor by precipitation with ammonium sulfate yielded a quite different and surprising result. Bacterial AHAS recovered its full activity, whereas the barley enzyme recovered none. Thus, inhibitor-induced inactivation of barley AHAS corresponds to a much less readily reversed change than in the case of bacterial enzyme. Experiments were carried out to explore whether enzyme inactivation required factors in addition to the inhibitor. Contrary to reports elsewhere, pyruvate (and therefore catalytic turnover) was not required. In the case of the bacterial enzyme, thiamine pyrophosphate (TPP)-Mg2/ was an absolute requirement. TPP bound some 220-fold more strongly to barley than to bacterial AHAS and, correspondingly, was also released a great deal more slowly (a half-time for dissociation of the enzyme:cofactor complex of Ç10 days compared to Ç1 hr). Here we suggest a mechanism whereby these differences in affinity for TPP-Mg2/ might underpin the apparent differences in the reversibility of sulfonylurea-induced inactivation of AHAS from the plant and bacterial sources. q 1996 Academic Press

INTRODUCTION

Acetohydroxyacid synthase (EC 4.1.3.18) (AHAS) catalyzes the first common step in the biosynthesis of branched chain amino acids (valine, leucine, and isoleucine) in enteric bacteria, yeasts, and plants (1–3). It catalyzes a dual reaction to condense either (i) two molecules of pyruvate to eliminate CO2 and form a-acetolactate for valine and leucine biosynthesis or (ii) one molecule of pyruvate and one molecule of aketobutyrate to eliminate CO2 and form a-acetoa-hydroxybutyrate for isoleucine biosynthesis. AHAS requires two cofactors to carry out these reactions: thiamine pyrophosphate (TPP) complexed to a divalent cation (Mn2/ or Mg2/) and flavine adenine dinucleotide (FAD). Given the 1 To whom correspondence should be addressed. Fax: (33) 04 68 66 22 26. E-mail: [email protected].

nonredox nature of the catalytic reaction, the requirement for flavin is intriguing (4). Based upon significant similarities in the protein sequence, it has been suggested that AHAS shares common ancestry with pyruvate oxidase, a TPPdependent flavoenzyme encoded by the pox B gene (5). The flavin and TPP sites of AHAS might thus be expected to lie adjacent to each other (cf. 6) and it has been speculated that evolution may have favored the retention of flavin purely in order to shield the nearby hydroxyethyl-TPP intermediate from protons (7). On the other hand, FAD may be more important in maintaining the quaternary structure of the enzyme (8). AHAS is a remarkable enzyme, being the target of several distinct classes of commercial herbicides including the sulfonylureas (9), the imidazolinones (10), the triazolopyrimidine

231 0048-3575/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.

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sulfonamides (11, 12), and pyrimidine oxybenzoates (13, 14). It has been speculated that this apparent susceptibility to inhibition by such a diverse set of chemistries relates to the partial retention of a quinone binding site originating from its pyruvate oxidase-related ancestry (7). Not surprisingly, a great deal of work has gone on to characterize the interactions of inhibitors with this important target for herbicide action. Inhibition of AHAS II from Salmonella typhimurium by sulfonylurea herbicides is time-dependent and is best described by a model in which an initial, relatively weak, enzyme/inhibitor (EI) complex slowly isomerized to a more tightly bound (EI*) form (15). The same model also appeared to adequately describe time-dependent inhibition of the pea enzyme by triazolopyrimidines, imidazolinones, sulfonylureas, and various other inhibitors (16). This being the case, it would be expected that inhibition should be reversible but only slowly so (17). For example, based upon an estimated Ki value for the initial complex, EI of 60 mM, a first-order rate constant of 0.05 min01 and a final K* i value for the EI* complex of 2 mM, Hawkes and Thomas (18) calculated an expected half-time of Ç6.5 hr for the dissociation rate of the presumptive EI* complex of imazapyr with AHAS from pea. Similarly, a half-time of Ç1.6 hr was calculated for the dissociation of the corresponding complex between sulfometuron methyl and AHAS II from S. typhimurium (19). However, it is notoriously difficult to arrive at accurate estimates for K* i and estimates of the dissociation rate of EI* should be taken as correspondingly uncertain. Two types of experiment are reported in the literature which have further explored the question of the reversibility of inhibition (i) measuring the physical binding of radiolabeled inhibitors to AHAS and (ii) measuring the rate of recovery of enzyme activity following removal of inhibitor. There is no obvious chemical rationale for expecting any of the inhibitors to bind irreversibly to protein via formation of a covalent

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bond. Studies with radiolabeled herbicides suggested that inhibitors bind reversibly to AHAS and that the enzyme/inhibitor complex dissociates relatively rapidly upon buffer exchange via gel filtration (15, 20, 21). Consistent with this, the inhibition of AHAS from S. typhimurium by sulfometuron methyl and by imazaquin (15, 22), from barley by a triazolopyrimidine sulfonamide (11, 12), from corn by imazapyr (18), and from Catharanthus roseus by sulfometuron methyl and by imazapyr (23) have all been described as reversible. In apparent contrast, Hawkes and Thomas (18) reported that AHAS in cultured carrot cells and in vitro from peas did not recover activity following exposure to either imazapyr or sulfometuron methyl. In the in vitro experiment, this ‘‘herbicide-induced inactivation’’ of the enzyme was only observed in the presence of pyruvate and TPP-Mg2/. It was noted that these factors had been absent from the experiments referred to above which had showed inhibition to be reversible. Durner et al. (21) obtained similar results following exposure of the corn enzyme to either chlorsulfuron or imazaquin. In this work it was further shown that repeat gel filtration of the inhibited complex led to substantial physical loss of radiolabeled inhibitor, but without any corresponding recovery of enzyme activity. On the basis of these results it was suggested (18, 21) that the initial reversible binding of inhibitors somehow triggers a slow shift to a stable and less active conformation of the enzyme. It was proposed that this process of inactivation rather than the supposed gradual isomerization of a reversible enzyme:inhibitor complex to a more stable form might fully account for the commonly observed time dependency of inhibition. However, it should be noted that probably both effects contribute. Certainly there is physical evidence that, at least in some cases, the eventually formed enzyme:inhibitor complex is indeed relatively stable and does not immediately dissociate upon buffer exchange. Radiolabeled chlorsulfuron remained in almost stoichiometric association with the barley enzyme following gel filtration (HPLC) of

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the enzyme in mixture with pyruvate, TPPMg2/, and the radiolabeled inhibitor (21). ‘‘Inhibitor-induced inactivation’’ is clearly an unusual phenomenon which must contribute to the unusual efficacy of the herbicides which act through inhibition of AHAS (24). However, there remains no convincing mechanistic explanation. Here we reexamine the somewhat vexed question of the reversibility of inhibition and, in so doing, compare and contrast the apparent inactivation of AHAS from barley by thifensulfurom methyl with inactivation of AHAS II from Escherichia coli. In particular, we address the unresolved question of whether catalytic turnover need be involved and propose a possible role for TPP-Mg2/. MATERIALS AND METHODS

Chemicals and chromatographic media. Reagent grade chemicals were from Sigma Chemical Co., except for L-tryptophan, which was purchased from Merck. Thifensulfuron methyl was supplied from Procida (RousselUclaf). [3H]Thifensulfuron methyl (3.14 TBq/ mmol) was synthesized as described previously (25). Phenylsepharose and PD-10 Sephadex G25 minicolumns were purchased from Pharmacia LKB. Media and buffers. Minimal medium for E. coli growth contained 7 g/liter K2HPO4 , 3 g/ liter KH2PO4 , 0.1 g/liter NaCl, 1 g/liter (NH4)2SO4 , and 0.1 g/liter MgSO4 and was adjusted to pH 7.0. All buffers contained 0.1 M potassium phosphate. For isolation of barley AHAS this was adjusted to pH 7.0; in the case of the E. coli enzyme, it was adjusted to pH 8.0. ‘‘Phosphate buffer’’ was the buffer with no additives. ‘‘Extraction buffer’’ contained 20 mM FAD, 10 mM EDTA, 1 mM DTT, and 20% (v/v) glycerol. ‘‘Buffer A’’ contained 0.5 mM TPP, 20 mM FAD, and 0.1 mM MgCl2 . ‘‘Buffer B’’ contained 20 mM FAD, 0.8 mM MgCl2 , 0.5 g/liter BSA, and 4% (v/v) glycerol. ‘‘PD-10 Sephadex G25column buffer’’ contained 20 mM FAD and 20% (v/v) glycerol.

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Partial purification of AHAS from barley. Barley (Hordeum vulgare L.) plants were grown on wet paper in the dark at 227C for 4 days. Shoots from the etiolated seedlings were harvested, washed with distilled water, and stored at 0207C for later use. Partial purification of AHAS from etiolated barley shoots was carried out at 47C. Shoots (100 g) were homogenized with 200 ml of extraction buffer. The homogenate was filtered through two layers of cheesecloth (100 mm). After addition of 1% (w/v) solid polyvinylpyrrolidone (15 min), the mixture was centrifuged at 25,000g for 10 min. The supernatant was brought to 23% saturation with ammonium sulfate (1 M) and then centrifuged at 25,000g for 10 min. The supernatant was loaded onto a 50-ml IBF25 column of phenylsepharose equilibrated with the buffer A containing Ç1.0 M ammonium sulfate. The column was then washed with starting buffer until the A280 nm value of the eluent had approached zero. Proteins were eluted with a linear gradient of 1 to 0 M ammonium sulfate (400 ml), and fractions containing AHAS activity were pooled and precipitated with 60% saturated ammonium sulfate. After equilibration for 1 hr, the precipitated protein was collected by low-speed centrifugation and redissolved in potassium phosphate buffer. The partially purified AHAS (sp act 0.025 U/mg) was stored and frozen at 0207C, under which conditions it maintained its activity for at least 2 weeks. Partial purification of AHAS II from recombinant E. coli. The E. coli strain JA 221 (rk0 mk/ trpE5 gal0 leu0 lac0 recA0) containing the pRL106 plasmid (genotype ilvG468 [IlvG/]) (26) was kindly provided by R. P. Lawther (University of South Carolina, Columbia, South Carolina). This strain was grown at 307C for 20 hr in a 1-liter fermentor containing minimal medium supplemented with glucose at a final concentration of 0.7%. L-tryptophan and ampicillin were added at concentrations of 15 and 50 mg/liter, respectively. Cells were collected by centrifugation (15,000g for 20 min) and resuspended in extraction buffer. Isolation and partial purifica-

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tion of AHAS II was carried at 47C or, as indicated, at ice temperature. Ice-cooled cells were sonicated (Heat Systems Ultrasonics Inc., W-375 Sonicator; 70% duty cycle) three times for 30 s and then for 1 min. The homogenate was then centrifuged at 10,000g for 30 min. The pellet was discarded and the supernatant brought to 60% saturation with ammonium sulfate. The solution was centrifuged at 18,000g for 10 min and the ammonium sulfate-precipitated pellet dissolved in potassium phosphate buffer and stored at 0207C. Samples were used within 2 weeks of storage. Specific activities of preparations varied between 0.5 and 1.5 U/mg. Enzyme assays. AHAS assays were normally carried out in final volume of 200 ml of buffer A containing 50 mM pyruvate and were at 377C unless otherwise stated. Assays were stopped with the addition of 20 ml of 6 N H2SO4 , and incubated at 607C for 10 min. The reaction product (acetoin) was then determined according to the method of Westerfeld (27). Then, 350 ml of 0.5% (w/ v) creatine in distilled water, 350 ml of 5% (w/v) a-naphthol in 2.5 N NaOH, and distilled water were added to a final volume of 1.22 ml. Solutions were mixed and heated to 607C for 15 min, and the absorbance of the colored complex measured at 530 nm. Assays were carried out in triplicate. Protein concentration. Protein concentrations were determined by the Coomassie brilliant blue binding assay (Bio-Rad) of Bradford (28) using BSA as a standard. Control experiment to verify that thifensulfuron methyl was not degraded during enzymatic assays. [3H]Thifensulfuron methyl (40 nM) was incubated with partially pure barley enzyme in assay solution for 2 hr at 207C. The reaction mixture was then separated by gel filtration with a PD-10 Sephadex G25 column into a protein fraction (2 ml) and a ‘‘retarded’’ fraction (13 ml). The latter was acidified to pH 5.0 with monobasic potassium phosphate buffer and quantitatively extracted with three washes (13 ml) of chloroform. This was concentrated and loaded onto a thin-layer

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silica chromatographic plate (Kieselgel 60F254 , 0.2 mm, MERCK) alongside untreated [3H]Thifensulfuron methyl and thifensulfuron as references. Compounds were eluted with ethyl acetate/acetic acid (99.5:0.5, v/v), and radioactivity was monitored using an automatic analyzer (LB283 BERTHOLD). Greater than 99.5% of the total radioactivity was recovered. Based on its unchanged chromatographic behavior, thifensulfuron methyl did not appear to be hydrolyzed by partially purified AHAS over the 2-hr reaction time. Activity recovery of barley AHAS treated with thifensulfuron methyl. Solutions (1.2 ml) of barley AHAS (20 1 1003 U) were preincubated at 207C for 2 hr in the absence (control) and presence (sample) of 40 nM thifensulfuron methyl in potassium phosphate buffer, buffer A, or buffer A plus 50 mM pyruvate. Then, 0.7 ml of each of these solutions was rapidly exchanged by gel filtration down a PD10 column of Sephadex G25 preequilibrated with ‘‘PD-10 Sephadex G25-column buffer’’ and further diluted into buffer A containing 50 mM pyruvate (2.0 ml). Assays of activity were then initiated (approximate zero time) by raising the temperature to 377C. Acetoin was determined in appropriate sized aliquots taken after 30, 60, 90, 120, and 170 min, respectively. Activity recovery of E. coli AHAS II treated with thifensulfuron methyl. Solutions (1.0 ml) of E. coli AHAS II (60 1 1003 U) were preincubated at 207C for 2 hr in the absence (control) and presence (sample) of 100 nM thifensulfuron methyl in buffer A, buffer A plus 50 mM pyruvate, potassium phosphate buffer plus 20 mM FAD and 50 mM pyruvate, or potassium phosphate buffer plus 0.5 mM TPP and 0.1 mM MgCl2 . Next, 0.5 ml of each of these solutions was then rapidly exchanged by gel filtration down a PD10 column of Sephadex G25 preequilibrated with ‘‘PD-10 Sephadex G25-column buffer’’ and further diluted into buffer A containing 50 mM pyruvate (2.0 ml). Assays of activity were then initiated (approximate zero time) by raising the temperature to 377C. Acetoin was determined in ap-

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propriate sized aliquots taken after 20, 30, 40, 60, 90, 120, 150, and 180 min, respectively. Inactivation of AHAS by thifensulfuron methyl and recovery of activity in ammonium sulfate precipitated protein. Barley AHAS (16.6 1 1003 U) and E. coli AHAS II (44.8 1 1003 U) were each incubated for 2 hr at 207C in 2 ml of buffer A containing 50 mM pyruvate and either 40 or 100 nM thifensulfuron methyl, respectively. Controls contained no inhibitor. Product formed over the 2-hr period was determined as acetoin in aliquots (150 ml for barley AHAS and 50 ml for E. coli AHAS II, respectively). The remainder of the two incubation mixtures was brought to 50% saturation with ammonium sulfate and allowed to stand 1 hr on ice. The protein pellets collected after low-speed centrifugation (15,000g for 10 min) were each then redissolved into an equal volume of buffer A containing 50 mM pyruvate. The activity of the redissolved proteins were then estimated as acetoin formation after 1 hr at 377C in 150 ml (barley AHAS) or 50 ml (E. coli AHAS II) aliquots of the two solutions. Time course of inhibitor induced AHAS inactivation: Influence of pyruvate. Barley AHAS (at a final concentration of 11 1 1003 U per ml) was incubated at 207C in buffer A either with or without 50 mM pyruvate and also in the absence (control) or presence of 40 nM thifensulfuron methyl. At 30, 60, 90, 120, 150, and 180 min, 0.5-ml aliquots were exchanged via gel filtration down a PD-10 column of Sephadex G25 into 1.5 ml of inhibitorfree buffer A containing 50 mM pyruvate. Aliquots (400 ml) of exchanged AHAS (2.9 1 1003 U) were then immediately raised to a temperature of 377C, and acetoin formation was measured after 1 hr. E. coli AHAS II (at a final concentration of 27 1 1003 U/ml) was incubated at 207C in buffer A either with or without 50 mM pyruvate and also in the absence (control) or presence of 100 nM thifensulfuron methyl. At 30, 60, 90, 120, 150, and 180 min, 0.5-ml aliquots were exchanged via gel filtration down a PD-10 column of Sephadex G25 into

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1.0 ml of inhibitor-free buffer A containing 50 mM pyruvate. Aliquots (200 ml) of exchanged AHAS (4.8 1 1003 U) were then immediately raised to a temperature of 377C and acetoin formation measured after 1 hr. Inhibitor-induced inactivation of AHAS II: Experiment to determine the influence of TPP. AHAS II (10 ml, 2.5 1 1001 U), 5 ml of 0.8 mM thifensulfuron methyl, and 5 ml of buffer B { 8 mM TPP were mixed. Controls were the same except without thifensulfuron methyl. These solutions were preincubated at 377C for 30 min and then diluted with the addition of 980 ml of buffer B. These were then incubated at 377C for an additional 5 min and then further diluted with 19 ml of potassium phosphate buffer. The activity of a 180ml aliquot was then determined over a relatively brief (30 min) assay period. Activation of E. coli AHAS II by TPP: Time course and dependence upon cofactor concentration. The TPP-dependent form of E. coli AHAS II (1.75 1 1003 U) was incubated with various concentrations of TPP (0, 10, 20, 40, 100, and 500 mM) in 1.5 ml buffer A containing 50 mM pyruvate at 377C. Aliquots (30 to 300 ml, depending upon the expected activity) were taken at 10- or 15-min intervals over a 135-min reaction time. Product formation was analyzed as acetoin as described above. Data processing. Data corresponding to Eq. (1) were fitted by a nonlinear regression analysis using the Deltagraph program (Midvision Inc.). RESULTS

Time-dependent inhibition of AHAS by thifensulfuron methyl. Thifensulfuron methyl inhibited AHAS II from E. coli and AHAS from barley in a similar time-dependent manner. The time courses of inhibition (not shown) were closely similar to those which have been described previously (cf. 15, 16, 21) for the inhibition of AHAS by other sulfonylurea herbicides and, similarly, could be modeled in terms of a slow first-order transition from an initial, relatively weak complex of the enzyme with inhibitor to a final, more potently inhib-

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ited form. Fitting data to this model yielded values comparable to those obtained in previous studies with first order transients governed by rate constants of 0.036 and 0.048 min01, respectively, for barley and E. coli AHAS II. Inhibition was mixed with respect to pyruvate concentration with apparent initial and final Ki values of 35 and 4.5 nM for barley enzyme and 102 and 8.5 nM for the E. coli enzyme. Reversibility of inhibition: Inhibitor-induced inactivation of AHAS. A premise of the biphasic model of inhibition is that it should be reversible. From the above data, the expected rates for dissociation of the putative tight complexes of thifensulfuron methyl with the two enzymes can be calculated as 0.0053 min01 (half-time of 130 min) for barley AHAS and 0.0044 min01 (half-time of 157 min) for E. coli AHAS II. It should be noted that although it is difficult to obtain accurate estimates for ‘‘final’’ Ki values from time courses such as those described above (i.e., at concentrations of inhibitor low enough to yield near 50% inhibition, the first-order transient becomes extended over many hours) our best estimates are close to those reported elsewhere (cf. 15, 18, 21). Indeed, even allowing for considerable error, some detectable recovery of enzyme activity would be expected within 3 hr of exchanging the inhibited enzyme into inhibitor-free buffer. However, the linearity of the time courses depicted in Figs. 1 and 2 show clearly that this was not the case. This was also confirmed (data not shown) in experiments in which the inhibited enzymes were directly diluted (rather than gel filtered) into inhibitor-free buffer. Thus, rather than reversible inhibition we observed ‘‘inhibitor-induced inactivation’’ of AHAS. Inhibitor-induced inactivation of AHAS: Recovery of activity after ammonium sulfate precipitation. Muhitch et al. (20) used ammonium sulfate precipitation to ‘‘reextract’’ fully active AHAS from enzyme inhibited with imazapyr. Thus we also tried precipitation of the protein with ammonium sulfate as an alternative method for separating inactivated enzyme from excess inhibitor. Somewhat surprisingly

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FIG. 1. Activity recovery after gel filtration of barley AHAS treated with thifensulfuron methyl. Barley AHAS was preincubated at 207C for 2 hr in the absence (control) (closed symbols) and presence (sample) (open symbols) of 40 nM thifensulfuron methyl in potassium phosphate buffer (j, h), buffer A (m, n), or buffer A plus pyruvate (l, s). Proteins were then rapidly exchanged by gel filtration down a PD10 column of Sephadex G25 and assayed at 377C for AHAS activity. Quantities of acetoin at each time point are expressed per 120-ml aliquot of assay solution. The activities of the controls for pre-incubation in the three different buffers in the absence of inhibitor were very similar and only the mean values (l) are plotted.

this procedure yielded significantly different results from gel filtration and direct dilution. As shown in Table 1, while barley AHAS still remained inactivated E. coli AHAS II activity recovered almost 80% of the control activity (note that in Table 1, the apparent difference between the control activities before (A) and after (B) precipitation with ammonium sulfate simply arises from the different conditions of temperature in the two phases of the experiment, i.e., 20 versus 377C). Inhibitor-induced inactivation: Requirement for substrate and cofactors. AHAS and thifensulfuron methyl were incubated in the presence of different combinations of either substrate or cofactors. Figure 1 shows that barley AHAS remained stably inactivated after removal of excess inhibitor and that this inactivation required neither pyruvate nor any other cofactor. Figure 2 shows that AHAS II from E. coli was similarly inactivated but that, unlike the barley enzyme, inactivation of the

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FIG. 2. Activity recovery after gel filtration of E. coli AHAS II treated with thifensulfuron methyl. E. coli AHAS II was preincubated at 207C for 2 hr in the absence (control) (closed symbols) and presence (sample) (open symbols) of 100 nM thifensulfuron methyl in buffer A (m, n), buffer A plus pyruvate (l, s), potassium phosphate buffer plus FAD and pyruvate (l, L), or potassium phosphate buffer plus TPP and MgCl2 (., ,). Proteins were then rapidly exchanged by gel filtration and assayed at 377C for AHAS activity. Quantities of acetoin at each time point are expressed per 100-ml aliquot of assay solution. The activities of the controls for preincubation in the different buffers in the absence of inhibitor were very similar and only the mean values (l) are plotted.

E. coli enzyme was conditional upon the presence of TPP-Mg2/ during the reaction with inhibitor. Where it was absent initially, the activity of AHAS II could be completely recovered after removal of thifensulfuron methyl. A further experiment to determine the

influence of TPP on the inactivation of AHAS II is detailed under Materials and Methods. Enzyme was preincubated for 30 min with inhibitor, separated from excess inhibitor by a 1000-fold dilution (rather than gel filtration) and then assayed for 1 hr. Consistent with Fig. 2, this experiment showed that in the initial absence of TPP-Mg2/, the activity of bacterial AHAS remained similar to the control, while in its presence, 30 min preexposure to 200 nM thifensulfuron methyl led to a 48% reduction in activity. Further similar experiments (data not shown) showed that TPP-Mg2/ was the only important factor and that pyruvate and FAD had no significant additional influence upon inactivation. Inhibitor-induced inactivation: Time course in the presence and in the absence of pyruvate. The experiments described in Fig. 3 show that the degree of inhibitor-induced inactivation of AHAS (as assayed by gel filtration to remove excess inhibitor and subsequent assay) slowly increased with time. The inactivation reaction was incomplete over the 3-hr period of the experiment. The ‘‘dead’’ time during gel filtration limited the accuracy of the experiment (the effect was somewhat minimized through the use of miniature columns and a relatively low temperature of 207C compared to 377C). The maximum level of inhibition looked to be tending toward a limiting value considerably less than 100% and to be associated with a

TABLE 1 Inactivation of AHAS by Thifensulfuron Methyl and Recovery of Activity in Ammonium Sulfate-Precipitated Protein

Enzyme

Enzymic assay

Barley AHAS E. coli AHAS II

A B A B

Controla activity (nmol/min/mg) 10 13.5 360 584.4

{ { { {

Residualb activity (nmol/min/mg)

0.5 0.4 7.2 45

4.2 5.8 134.1 453.6

{ { { {

1 0.3 2.7 31.5

Inactivation (%) 57.5 57.0 62.8 22.4

a

{ { { {

2.4 2.1 1.2 1.6

Controls contained no inhibitor. Barley AHAS and E. coli AHAS II were each incubated for 2 hr at 207C in buffer A containing pyruvate and either 40 or 100 nM thifensulfuron methyl, respectively. (A) Product formed over the 2-hr period was determined as acetoin in aliquots. (B) The activity of ammonium sulfate precipitated protein was estimated as acetoin formation in aliquots after 1 hr at 377C in buffer A containing pyruvate. b

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Time course of AHAS activation as a function of TPP concentration. We investigated the activation of E. coli AHAS II by TPP. The data of Fig. 4 fit the model described previously for barley AHAS (29), suggesting that the activation process involves two slow steps. The rate constants in Scheme 1 were determined according to Eq. [1] (Table 2), where P is product, Po is product present initially, Ves is the final steady-state rate, and k is the pseudo-first-order rate constant of activation. P Å Vesrt 0 (Ves/k)r(1 0 e0krt) / Po ;

[1]

k Å (k1/k2)r(k3 / k4)r[TPP] / k4)/(k1/k2) 1 [TPP] / 1) k1

k3

E / TPP S E-TPP S E-TPP* k2

k4

SCHEME 1. Activation by TPP of AHAS. FIG. 3. Time course of inhibitor-induced AHAS inactivation: Influence of pyruvate. (A) Barley AHAS and (B) E. coli AHAS II were incubated at 207C in buffer A either with (l) or without (s) pyruvate in the absence (control) or presence of 40 and 100 nM thifensulfuron methyl, respectively. At 30, 60, 90, 120, 150, and 180 min, aliquots were exchanged via gel filtration into inhibitor-free buffer A containing pyruvate. Acetoin formation at 377C was measured after 1 hr. Results are % inhibition.

time dependence broadly similar to that usually described (i.e., see above and 15, 18, 21) for slow biphasic inhibition of AHAS. Including pyruvate in the experiment (and therefore having the enzyme in a catalytically active state) clearly had no influence on either the extent or the rate of inactivation. AHAS activity as a function of TPP concentration. In the absence of added TPP, bacterial AHAS II exhibited only 4% of the activity at saturating (500 mM) TPP concentrations. Our preparation of barley AHAS appeared, on the other hand, to have substantially copurified with the cofactor since its activity assayed in the absence of TPP was more than 80% of the activity in its presence.

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The parameters for TPP activation reveal a large difference between plant AHAS and bacterial AHAS. The value of the affinity constant Ks is 10.5 mM for E. coli AHAS II, while the value reported for barley AHAS is 48 nM,

FIG. 4. Time course of activation by TPP of AHAS II. AHAS II TPP-apoenzyme was incubated at 377C in buffer A containing pyruvate and 10 mM (j), 20 mM (l), 40 mM (s), 100 mM (m), and 500 mM (h) TPP. Aliquots were taken at 10, 20, 30, 45, 60, 75, 90, 105, 120, and 135 min, and acetoin was determined. Results correspond to a 150-ml reaction volume.

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TABLE 2 Kinetic Parameters Governing the Activation of AHAS II by TPP

Enzyme E. coli AHAS II Barley AHAS (29)

k4 , 103 (min01)

(k3 / k4), 103 (min01)

k3 , 103 (min01)

12.3

170.4

158.1

93.0

93.0

0.04

Ks (mM) 10.5 0.048

k2 /k1 (mM) 148.4 112.0

Note. Values are calculated from data from Fig. 4 according to Eq. [1].

under conditions that were identical to the ones we used for our studies (29). The affinity of AHAS II for TPP is therefore some 220fold less than for barley AHAS. It can be seen that the difference in the overall affinity is due mainly to the difference in dissociation rates k4 . All the other parameters fall into the same range of values between the two enzymes. From the value of k4 , the half-time for dissociation of the AHAS II/TPP complex can be estimated at only 56 min, in contrast to more than 10 days (29) for the complex of TPP with the barley enzyme. DISCUSSION

Thifensulfuron methyl inhibited AHAS II from E. coli and AHAS from barley in a timedependent manner. In good agreement with earlier studies of the inhibition of AHAS (cf. for inhibition of the pea enzyme by sulfometuron methyl, 16) we found that slow inhibition could be modeled (15) in terms of a slow firstorder isomerization (Ç0.04 min01) governing an apparent Ç10-fold reduction in the apparent Ki value of the enzyme inhibitor complex. However, the mere fitting of progress curves need not imply that the model used to describe the data be correct. Implicit in the biphasic model is the expectation that inhibition should be reversible. However, we saw no sign of the expected slow recovery of enzyme activity following exchange (by gel filtration or dilution) of the inhibited barley (Fig. 1) or E. coli (Fig. 2) enzymes into inhibitor-free buffer. Thus, similar to the previously observed inactivation of pea AHAS by imazapyr or by sul-

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fometuron methyl (18) and of corn AHAS by chlorsulfuron (21), inhibition of barley AHAS by thifensulfuron methyl also appeared, under the conditions of our experiment, irreversible. Durner et al. (21) showed that corn AHAS remained inactivated even after dissociation of the enzyme/inhibitor complex and coined the phrase ‘‘inhibitor-induced enzyme inactivation’’ to describe the phenomenon. We carried out further experiments to further explore the nature of inhibitor-induced inactivation. As expected (Fig. 3) the degree of enzyme inactivation increased with the time of reaction with inhibitor in such a way as would account for what has previously been described as time-dependent ‘‘inhibition.’’ Given that (i) time-dependent inhibition has been said to be ‘‘pyruvate-dependent’’ (15) and (ii) that inhibitor-induced inactivation of pea AHAS did not occur in the absence of TPP-Mg2/ and pyruvate (18), it has been implicit that enzyme turnover might somehow be important in the mechanism of inhibitorinduced inactivation. A very important finding of the present work is that this is not the case. The data in Figs. 1–3 all clearly show that the presence or absence of pyruvate had no bearing at all on the either the rate or extent of inactivation of AHAS following exposure to herbicide. This effectively rules out what has previously looked to be one of the more convincing proposals put forward to explain AHAS inactivation (30), that inhibitors might somehow increase the tendency of the hydroxyethyl thiamine pyrophosphate (HETPP) intermediate toward reaction with molecular ox-

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ygen and inactivation via formation of the hydroperoxide. In the absence of pyruvate there would be no HETPP to undergo this reaction. The sole additional requirement for the inactivation of AHAS II from E. coli by thifensulfuron methyl was TPP-Mg2/. For the barley enzyme not even this was required. However, given that TPP was neither required for the activity of the barley enzyme it was clear that our preparation must have retained nearstoichiometric amounts of cofactor. Thus all the data were consistent with TPP-Mg2/ being the only essential corequirement for inhibitorinduced inactivation of AHAS. Is TPP-Mg2/ required to create the binding site for inhibitors (it certainly is for substrates) or do inhibitors bind anyway but only with TPP in situ does the enzyme slowly become inactivated? While radiolabeled inhibitors have been shown to bind to AHAS in the absence of added TPP (15, 20), the content of TPP in enzyme preparations has not always been defined. Since addition of the sulfonylurea herbicide sulfometuron methyl to isozyme II of AHAS from S. typhimurium considerably slowed the exchange rate of TPP-Mg2/ bound to the enzyme (22), it would seem that, in reciprocation, sulfonylurea inhibitors must bind more strongly to the enzyme TPP-Mg2/ complex than to the apoenzyme. This view is further strongly supported by the observation of Schloss et al. (7) that the stoichiometry of sulfometuron methyl binding to the Salmonella enzyme closely paralleled the content of TPP-Mg2/ in the particular preparation. Schloss et al. (7) used equilibrium dialysis to arrive at a Kd of 14 mM for the dissociation constant of the complex of radiolabelled sulfometuron methyl with the Salmonella AHAS:TPP-Mg2/ complex. These workers ascribed the discrepancy between this and their estimate of the final inhibition constant (0.08 mM ) as most likely due to the influence of pyruvate on promoting tighter binding of the inhibitor during enzyme turnover. However, we have shown here that the estimated final inhibition constant would, in reality,

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have arisen from gradual enzyme inactivation, that pyruvate would have had no influence on this process, and therefore that the dissociation constant arrived at by equilibrium dialysis does perhaps represent the true maximum strength of binding of this class of inhibitors. Having established that TPP-Mg2/-dependent binding of thifensulfuron methyl to AHAS caused some long-term inactivation of the enzyme, we carried out experiments to further study the reversibility of the effect. While not, apparently, reversed by buffer exchange or dilution into inhibitor-free buffer it was immediately surprising to note that almost the full activity of AHAS II from E. coli could be recovered if the enzyme were recovered from inhibition by precipitation with ammonium sulfate (Table 1). The same was not true of barley AHAS. This indicated (i) that inhibitor-induced inactivation could, at least in principle, be reversed (and that whatever process it corresponds to must therefore be unlikely to involve any covalent modification to the protein itself) and (ii) that there was some significant difference in the inactivation of the E. coli and barley enzymes. A clue to what this significant difference might be was revealed by our comparative studies of the requirement for and activation by TPP-Mg2/ of the two AHAS enzymes. While our preparation of barley AHAS exhibited 80% of its full activity without the addition of TPP-Mg2/ to the assay solution, the residual activity of AHAS II under the same conditions was only 4% of the control. The detailed kinetic analysis of activation of AHAS II by TPP further revealed an overall affinity constant, Ks , of 10.5 mM compared to 48 nM for AHAS from barley (29). TPP-Mg2/ is much more tightly bound to barley AHAS (t1/2 dissociation ú 10 days) than to isozyme II of AHAS from E. coli. (t1/2 dissociation Å 56 min), thus explaining why the plant enzyme should retain the cofactor during purification. As regards the mechanism of herbicideinduced inactivation, there are, at the molec-

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ular level, many possible ways by which some quasi-stable shift in protein conformation might impact upon catalytic activity. Here we have highlighted the vital role of TPP-Mg2/ in this process. We have shown that (i) TPP-Mg2/ is the sole and absolute corequirement for inhibitor-induced inactivation of AHAS and (ii) that the relative ‘‘permanence’’ of inactivation suffered by enzymes from different sources is likely to be linked to differences in the exchange rate of the protein-bound cofactor. Thus, we suggest that while the herbicide provides the ‘‘trigger’’ for inactivation, TPP-Mg2/ might provide the ‘‘lock’’ with the stability of herbicide-induced inactivation directly relating to the tightness of TPP-Mg2//AHAS interactions after the removal of the inhibitor. The relative affinity of AHAS from different origins for TPP-Mg2/ probably varies quite widely (for example, Schloss (22) observed an exchange rate for TPP-Mg2/ bound to the isozyme II of AHAS from S. typhimurium some 10-fold more rapid than observed here for the enzyme from E. coli ) and the stability of herbicide-induced inactivation might thus be expected to vary accordingly. Speculating further on how changes in the binding of TPP-Mg2/ might directly reduce the activity of the enzyme it is possible that the cofactor itself assumes a less catalytically efficient conformation. For example, Schneider and Lindqvist (31) proposed a mechanism for the thiaminic catalysis of transketolase (EC 2.2.1.1) involving the formation of a tautomeric 4*-imino group as the C(2)-H proton acceptor of the thiazolium ring. Inhibitor-induced inactivation of AHAS might then correspond to a shift in protein conformation disfavouring the binding of TPP in the ‘‘V’’ conformation necessary for this specific internal catalytic interaction. ACKNOWLEDGMENT We thank Dr Ronald L. Somerville for his thoughtful comments and corrections to this manuscript.

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