Aryloxyphenoxypropanoate and ... - ACS Publications

Chapter 18

Aryloxyphenoxypropanoate and Cyclohexanedione Herbicides Inhibition of Acetyl Coenzyme A Carboxylase Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch018

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Jacob Secor , Csaba Cséke , and W. John Owen 1

Agricultural Products Research, Dow Chemical Company, Walnut Creek, CA 94598-0902 Department of Biochemistry, Royal Holloway and Bedford New College, Egham Hill, Surrey, TW20 OEX, England 2

Haloxyfop, an aryloxyphenoxypropanoate, and tralkoxydim, a cyclohexanedione, inhibit acetyl coenzyme A carboxylase (ACCase) from moncotyledoneous and dicotyledoneous species in a manner indicating that this enzyme is the target site for these classes of herbicides. The concentration of haloxyfop and tralkoxydim required for 50% inhibition (I ) of ACCase activity in extracts from susceptible species (maize, wheat, and t a l l fescue) ranged from 0.5 μΜ to 1.2 μΜ. The I values for a resistant monocot, red fescue, were 10 μΜ and 30 μΜ, and for a resist ant dicot, soybean, were 140 μΜ and 520 μΜ for haloxyfop and tralkoxydim, respectively. The I values correlate very well with the herbicidal activity of theses compounds except for wheat, which is resistant to tralkoxydim. The herbicidally in active S(-) enantiomer of haloxyfop did not inhibit maize ACCase. Aryloxyphenoxypropanoates and cyclohexanediones are two classes of herbicides that control many monocotyledoneous species. Although these herbicides are structurally very different (Fig. 1), there has been some conjecture that they have a similar mode of action because of their similarity in selectivity and symptomology. This paper describes the experiments that led to the discovery that aryloxyphenoxypropanoate and cyclohexanedione herbicides inhibit acetyl coenzyme A carboxylase (acetyl-coenzyme A: bicarbonate ligase [ATP], EC 6.4.1.2) activity in susceptible species (1). In addition, evidence is presented indicating that the inhibition of acetyl coenzyme A carboxylase (ACCase) is well correlated to observed herbicidal activity. Similar, independent findings have recently been reported by two other research groups (2.3). 50

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0097-6156/89/0389-0265$06.00/0 © 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch018

Mode of Action Since the discovery of the h e r b i c i d a l properties of the aryloxyphenoxypropanoates, there have been many studies aimed at deter mining t h e i r mode of action. Fewer reports have been published regarding the mode of action of cyclohexanediones. Neither class of compounds interferes with photosynthesis, respiratory O2 uptake, protein biosynthesis, or nucleic acid biosynthesis (4-6). Several physiological processes are disrupted by both the cyclo hexanediones and aryloxyphenoxypropanoates, namely growth and development, maintenance of membrane i n t e g r i t y , auxin induced growth, and l i p i d metabolism (4.5.7.8). In addition, the aryloxy phenoxypropanoates have been reported to depolarize membrane potentials (9.10). However, no s p e c i f i c target s i t e had been i d e n t i f i e d for either class of compounds. Of the postulated modes of action of these compounds, the i n h i b i t i o n of l i p i d biosynthesis has been most thoroughly inves tigated, p r i n c i p a l l y by Hoppe and colleagues (4.5.11.12) for the aryloxyphenoxypropanoates and Lichtenthaler and colleagues (13.14) for the cyclohexanediones. Hoppe's group demonstrated f i r s t that the aryloxyphenoxypropanoate dielofop-methyl (methyl 2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate) altered l i p i d metabolism (4). They went on to show that acetate incorporation into f a t t y acids was i n h i b i t e d a f t e r a 60 min incubation i n diclofop methyl i n chloroplasts of susceptible but not tolerant species (11). The cyclohexanedione sethoxydim (2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one) also alters lipid metabolism by i n h i b i t i n g acetate incorporation into f a t t y acids (14). These studies strongly suggested that de novo f a t t y acid synthesis was i n h i b i t e d but did not i d e n t i f y the s p e c i f i c s i t e of inhibition. Experiments Leading to ACCase Our goal was to determine the s p e c i f i c target s i t e ( s ) of action of the aryloxyphenoxypropanoate and cyclohexanedione herbicides. We set two simple c r i t e r i a as prerequisites for i d e n t i f y i n g physio l o g i c a l processes as possible target s i t e s . The s i t e of action must be affected (1) rapidly (within minutes) and (2) by low concentrations (760 133 225 1250 >6000 >6000 >6000 50

Species Maize Wheat T a l l fescue Red fescue Soybean

ISO (μΜ) Haloxyfop Tralkoxydim 0.52 0.50 0.91 1.22 0.40 0.94 13.83 23.32 516.72 138.50

R e v e r s i b i l i t y of Binding of ACCase and the

Herbicides

Once that i t was established that ACCase was a target s i t e of the aryloxyphenoxypropanoates and cyclohexanediones, we began to investigate the relationship between the herbicides and the enzyme. Using a protein extract prepared as previously described (1) and further p u r i f i e d through a Sephacryl S-300 gel f i l t r a t i o n column, we determined whether the i n h i b i t o r s were covalently bound


18.

SECOR ET AL.

Herbicidal Inhibition ofAcetyl Coenzyme A Carboxylase

to the enzyme. ACCase was incubated i n a 7 μΜ herbicide solution and then assayed before and after passing through a desalting column (Sephadex G-25). As expected, the herbicides i n h i b i t e d ACCase a c t i v i t y (Table V). After passage through the desalting column, ACCase was no longer i n h i b i t e d by the herbicides. These results indicate that the herbicides are not covalently bound to the enzyme.


Table V. Effect of Desalting an ACCase-Herbicide Mixture on Enzyme A c t i v i t y

Treatment 15 min incubation at 35 C Desalting Haloxyfop1 Tralkoxydim 1

Enzyme A c t i v i t y (nmol/min/mg) Tralkoxydim^ Haloxvfopl 64 72 169 185 -

Control 254 168 43 31

^-Herbicide concentration i n assays was 1.4 μΜ.

There are many other questions that need to be addressed. For example: What are the k i n e t i c s of the inhibition? Do the d i f f e r e n t i n h i b i t o r s bind at the same site? What are the molecular require ments f o r inhibition? What are the differences between susceptible and tolerant ACCases? and so on. ACCase p u r i f i e d 40 to 100 f o l d may not be s u f f i c i e n t l y pure to answer many of these questions. For example, an extract p u r i f i e d on a Sephacryl S-300 column can have a s p e c i f i c a c t i v i t y up to 400 nmol/min/mg. We have observed that this preparation can catalyze the carboxylation of other short chained acyl CoA's i n addition to acetyl CoA (Table VI). Both haloxyfop and tralkoxydim i n h i b i t the carboxylation reaction regardless of whether n-propionyl CoA or acetyl CoA are substrates either i n d i v i d u a l l y or together (Table V I I ) . At present, we are unsure whether n-propionyl CoA can be used as a substrate f o r ACCase or whether a n-propionyl CoA carboxylase i s present i n the preparation and the herbicides also i n h i b i t that enzyme.

Table VI. Carboxylation of Different Acyl CoA Molecules Substrate (0.3 mM) Acetyl CoA n-Propionyl CoA η-Butyl CoA Isobutyl CoA V a l e r y l CoA

Reaction Rate

(nmol/min/mg)

192 59 32 0 1


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Aryloxyphenoxypropanoate and ... - ACS Publications

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