**Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides**

### E. A. Saratovskikh

[81] Ishimitsu S, Kaihara A, Yoshii K, Tsumura Y, Nakamura Y, Tonogai Y. Determina‐ tion of Clethodim and Its Oxidation Metabolites in Crops by Liquid Chromatogra‐ phy with Confirmation by Lc/Ms. Journal of AOAC International 2001;84(4)

[82] Klein J, Alder L. Applicability of Gradient Liquid Chromatography with Tandem Mass Spectrometry to the Simultaneous Screening for About 100 Pesticides in Crops.

[83] Gomyo T, Ono S. Residue Analysis of Herbicide Sethoxydim. Journal of Pesticide Sci‐

[84] Gomyo T, Kawakami H, Tokieda M, Sugioka K, Kobayashi S, Ono S. Residue Analy‐ sis of Herbicide Sethoxydim and Its Metabolites in Crops. Journal of Pesticide Sci‐

[85] Hu J-Y, Aizawa T, Magara Y. Analysis of Pesticides in Water with Liquid Chroma‐ tography/Atmospheric Pressure Chemical Ionization Mass Spectrometry. Water Re‐

[86] Eisert R, Jackson S, Krotzky A. Application of on-Site Solid-Phase Microextraction in Aquatic Dissipation Studies of Profoxydim in Rice. Journal of Chromatography A

[87] Tsochatzis ED, Tzimou-Tsitouridou R, Menkissoglu-Spiroudi U, Karpouzas DG, Ma‐ ria Papageorgiou G. Development and Validation of an Hplc-Dad Method for the Si‐ multaneous Determination of Most Common Rice Pesticides in Paddy Water Systems. International Journal of Environmental Analytical Chemistry 2012;92(5)

[88] Liska I, Brouwer ER, Ostheimer AGL, Lingeman H, Brinkman UAT, Geerdink RB, Mulder WH. Rapid Screening of a Large Group of Polar Pesticides in River Water by on-Line Trace Enrichment and Column Liquid Chromatography. International Jour‐

[89] Shen J-c, Ye W-x, Xie L-q, Zhao Q-h, Xiao L-l. Analysis of Cyclohexanedione Herbi‐ cides in Rice and Corn by Lc-Ms/Ms. Journal of Chinese Mass Spectrometry Society

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130 Herbicides - Advances in Research

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54679

### **1. Introduction**

"A lack of knowledge in the area of biology of grown plants and specific features of the medium of their dwelling in each specific field cannot be compensated by an excess of pesticides, fertilizers, or melioration." Academician D.N. Pryanishnikov, 1934

Pesticides as a whole and herbicides in particular are substances with high biological activity. They can exert a toxic effect on many components of cells: enzymes, structural and functional proteins, lipoproteids, polysaccharides, nucleic acids, and others. The elucidation of the mechanism of toxic effect is an important challenge, the solution of which would allow one to establish the real and potential danger of application of these or other compounds for human and non-targent organisms. Despite the enormous scale of production and use of chemical means for cultivated plant protection, there is still much unknown on the mechanism of their action. It is considered that, probably, each pesticide acts through a unique mechanism. For example, the acting components of pesticides, namely, zenkor, lontrel, roundup, kusagard, setoxidim, basagran, tilt, and tachigaren, belong to different classes of chemical compounds. According to available literature data (Table 1), they interact with various enzymatic systems, have their own specific binding sites, and are characterized by different mechanisms of action.

Much data concerning the influence of herbicides and fungicides on various components of the living cell (Fedtke, 1982; Kadyshev, 1970; Fudel-Osipova, 1981), in particular, on some enzymes (Mathew et al., 1998; Forthoffer et al., 2001; Banas et al., 2000; Knecht & Löffler, 1998; Du, 2000a; Gruys et al., 1993; Nosanchuk et al., 2001; Kiyomiya et al., 2000) have been reported. For instance, anticholinesterase compounds, organophosphorus pesticides, carba‐ mates and triazines (Grin & Goldberger 1968), are structurally similar to substrates and

© 2013 Saratovskikh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Saratovskikh; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

competitively inhibit their activity. The effect was evaluated for fries of Mediterranean fishes *Dicentrarchus labrax* (Varo et al., 2003) and for rats (*Maple amber, M. arrow*) fed with soybean after treatment with zenkor and atrazine (Mathew et al., 1998). It was shown that herbicide basagran suppresses the antiphosphatecholinesterase activity and results in an increase in the hydroxylase activity (Al-Меndоfi & Ashton, 1984; Forthoffer et al., 2001).

The growth of fungi *Cryptococcus neoformans* was suppressed by glyphosate due to the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (Nosanchuk et al., 2001). A nonproductive four-membered complex is formed between the enzyme, pesticide, and phosphate (Du, 2000b). Octahedral coordination is performed by the metal ion: Co glyphosate enzyme as in 3-deoxy-D-arabiheptulosonate-7-phosphate synthase localized in cytosols (Ganson & Jensen, 1988).

Oxidative phosphorylation is performed by Zn-containing enzymes. Dinoseb, pentachloro‐ phenol, dichlorodiphenyltrichloroethane, and Sevin separate oxidative phosphorylation in mitochondria of Palma Christi (Kuz`minskaya, 1975) and decrease the ATP content in glycols of soybean (Gruenhagen & Моreland, 1971). Chloro-containing organic pesticide endosulfan reacts with glutathione (cofactor of glutathione peroxidase), considerably decreasing the activity of the enzyme. The loss of secretory reactions in thylakoids of adrenocortical steroi‐ dogenic cells and changes in the enzyme activity indicate that the pesticide was involved in the oxidative reactions (Dorval et al., 2003).

The formation of complexes of vegetable peroxidase with various substrate-inhibitors was established (Ugarova & Lebedeva, 1978). Both the direct participation of the metal in the substrate addition to the protein part of the molecule and providing of a relationship between the flavine group and apoenzyme under the action of the metal are assumed. The neighbor‐ hood of the pyridine nitrogen atom to the carboxyl group in picolinic acid (picloram) is manifested in the ability to complexation and metal removal from enzymes (Shcheglov et al., 1967).

Bagirov et al., 1989; Lycholat & Bilchuk, 1998) possessing a broad substrate specificity. This enzyme is in the composition of the monooxygenase system that utilizes substrates and transforms xenobiotics into the lowly toxic state. NADH-OR, [EC 1.6.99.25] from the methyl‐ otroph *Methylococcus capsulatus* (strain M) (Burbaev et al., 1990) transfers electrons for the mixed reduction of oxygen to water, methane transformation to give methanol in the active center of methane hydroxylase, and the reduction of dioxygen to water in the active center of cytochrome oxidase. The enzyme studied consists of four subunits, each including FAD and the iron-sulfur cluster, 2Fe-2S (Tsuprun et al., 1987; Bagirov et al., 1989). NADH-OR functions

(mitochondrial)

**Common name Range of application Mechanism action Reference**

a wide spectrum of action similar to auxin Hall et al., 1985

phosphate synthase

inhibitor of dehydrogenase

7-etoxyrezofurine O-diethylase; inductor glutathione S-transferase

a wide spectrum of action inhibitor of NADH-oxidoreductase Saratovskikh, 2005;

selective to grains inhibitor of photo- ; protein- ; lipids- ; RNA - synthesis

inhibitor of enolpyruvateshekemate-

complexes with membrane lipids Ziegler et al., 1982

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

lesion meristem tissues Iwataki & Hirono, 1978

Amrhein et al., 1980

http://dx.doi.org/10.5772/54679

133

Trebst & Wietoska,

Osama & Ashton, 1984

Knecht & Löffler, 1998

Levine & Oris, 1999; Egaas et al., 1999

Saratovskikh, 2007

1975

selective to dicotyledons and solanaceous

for struggle against perennial weeds

beet, solanaceous

selective to the grass, sugar

Kusagard selective to dicotyledons, beet, cotton

Setoxidim selective to dicotyledons,

beet

**Table 1.** Pesticides mechanism action from literary

The most part of biological oxidation processes is performed by an array of carriers, which are grouped in the electron transfer chain and the respiratory chain, one end of which contains the active metabolite and the 1/2О2–H2О system is localized on another end. Among the main components of the chain electron transfer are nicotinamide (pyridine) coenzymes NADH and

The sequence of electron transfer from NADH to an electron acceptor is still unknown. However, by analogy with other reductases, one can suggest that the electrons are transferred

according to the following scheme:

NAD<sup>+</sup> <sup>+</sup> *<sup>A</sup>*red

NADH-*ОR*

*NАDН* + *Aох* →

Zenkor, Metribuzin

Lontrel, Clopyralid

Roundup, Glyphosate

Basagran, Bentazon

Tachigaren, Hymexazol

Propiconazole

Lontrel metal complexes

Titl,

NADFH.

where A is acceptor.

The tests on human and rat tissues showed that tachigaren and its metabolites (four enzymes synthesizing pyrimidine) inhibit mitochondrial [ЕС 1.3.99.11]. This results in the changes in the pyridine–nucleotide pool that provides the work of immune cells. The reaction is reversible and its mechanism is uncompetitive with respect to the substrate and cofactor ubiquinone (Knecht & Löffler, 1998).

On the other hand, diverse xenobiotics, both pesticides and metals, are abundant in consider‐ able amounts in the nature, namely, in air, soil, and water (Banas et al., 2000; Knecht & Löffler, 1998; Du, 2000a). If these xenobiotics get into the human organism, they may cause various diseases (Gruys et al., 1993; Nosanchuk et al., 2001). In the presence of pesticides with ligand properties, their combined effect on living organisms can be enhanced or weakened.

The ability of the environment to self-purification, *i.e.*, decomposition of contaminants, is determined, to a great extent, by the occurrence of enzymatic redox processes in cells of plants and microorganisms. One of the enzymes performing the redox processes in biological systems is NАDН-oxidoreductase (NАDН-ОR) (Tukhvatullin et al., 2001; Sommerhalter et al., 2004;


**Table 1.** Pesticides mechanism action from literary

Bagirov et al., 1989; Lycholat & Bilchuk, 1998) possessing a broad substrate specificity. This enzyme is in the composition of the monooxygenase system that utilizes substrates and transforms xenobiotics into the lowly toxic state. NADH-OR, [EC 1.6.99.25] from the methyl‐ otroph *Methylococcus capsulatus* (strain M) (Burbaev et al., 1990) transfers electrons for the mixed reduction of oxygen to water, methane transformation to give methanol in the active center of methane hydroxylase, and the reduction of dioxygen to water in the active center of cytochrome oxidase. The enzyme studied consists of four subunits, each including FAD and the iron-sulfur cluster, 2Fe-2S (Tsuprun et al., 1987; Bagirov et al., 1989). NADH-OR functions according to the following scheme:

*NАDН* + *Aох* → NADH-*ОR* NAD<sup>+</sup> <sup>+</sup> *<sup>A</sup>*red

where A is acceptor.

competitively inhibit their activity. The effect was evaluated for fries of Mediterranean fishes *Dicentrarchus labrax* (Varo et al., 2003) and for rats (*Maple amber, M. arrow*) fed with soybean after treatment with zenkor and atrazine (Mathew et al., 1998). It was shown that herbicide basagran suppresses the antiphosphatecholinesterase activity and results in an increase in the

The growth of fungi *Cryptococcus neoformans* was suppressed by glyphosate due to the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (Nosanchuk et al., 2001). A nonproductive four-membered complex is formed between the enzyme, pesticide, and phosphate (Du, 2000b). Octahedral coordination is performed by the metal ion: Co glyphosate enzyme as in 3-deoxy-D-arabiheptulosonate-7-phosphate synthase localized in cytosols (Ganson &

Oxidative phosphorylation is performed by Zn-containing enzymes. Dinoseb, pentachloro‐ phenol, dichlorodiphenyltrichloroethane, and Sevin separate oxidative phosphorylation in mitochondria of Palma Christi (Kuz`minskaya, 1975) and decrease the ATP content in glycols of soybean (Gruenhagen & Моreland, 1971). Chloro-containing organic pesticide endosulfan reacts with glutathione (cofactor of glutathione peroxidase), considerably decreasing the activity of the enzyme. The loss of secretory reactions in thylakoids of adrenocortical steroi‐ dogenic cells and changes in the enzyme activity indicate that the pesticide was involved in

The formation of complexes of vegetable peroxidase with various substrate-inhibitors was established (Ugarova & Lebedeva, 1978). Both the direct participation of the metal in the substrate addition to the protein part of the molecule and providing of a relationship between the flavine group and apoenzyme under the action of the metal are assumed. The neighbor‐ hood of the pyridine nitrogen atom to the carboxyl group in picolinic acid (picloram) is manifested in the ability to complexation and metal removal from enzymes (Shcheglov et al.,

The tests on human and rat tissues showed that tachigaren and its metabolites (four enzymes synthesizing pyrimidine) inhibit mitochondrial [ЕС 1.3.99.11]. This results in the changes in the pyridine–nucleotide pool that provides the work of immune cells. The reaction is reversible and its mechanism is uncompetitive with respect to the substrate and cofactor ubiquinone

On the other hand, diverse xenobiotics, both pesticides and metals, are abundant in consider‐ able amounts in the nature, namely, in air, soil, and water (Banas et al., 2000; Knecht & Löffler, 1998; Du, 2000a). If these xenobiotics get into the human organism, they may cause various diseases (Gruys et al., 1993; Nosanchuk et al., 2001). In the presence of pesticides with ligand

The ability of the environment to self-purification, *i.e.*, decomposition of contaminants, is determined, to a great extent, by the occurrence of enzymatic redox processes in cells of plants and microorganisms. One of the enzymes performing the redox processes in biological systems is NАDН-oxidoreductase (NАDН-ОR) (Tukhvatullin et al., 2001; Sommerhalter et al., 2004;

properties, their combined effect on living organisms can be enhanced or weakened.

hydroxylase activity (Al-Меndоfi & Ashton, 1984; Forthoffer et al., 2001).

Jensen, 1988).

132 Herbicides - Advances in Research

1967).

(Knecht & Löffler, 1998).

the oxidative reactions (Dorval et al., 2003).

The most part of biological oxidation processes is performed by an array of carriers, which are grouped in the electron transfer chain and the respiratory chain, one end of which contains the active metabolite and the 1/2О2–H2О system is localized on another end. Among the main components of the chain electron transfer are nicotinamide (pyridine) coenzymes NADH and NADFH.

The sequence of electron transfer from NADH to an electron acceptor is still unknown. However, by analogy with other reductases, one can suggest that the electrons are transferred from NADH to FAD and then to the iron sulfur 2Fe-2S cluster and to the electron acceptor. Neotetrazolium chloride (NT) was used in this work as the artificial electron acceptor.

Enzymes of this type are present in the cells of almost all organisms. Therefore, the general features of the interaction of this enzyme with pesticides can also be applied to NADH-OR from other organisms.

[ <sup>C</sup>

+ +

ML2 where L is lontrel, were synthesized by refluxing ethanolic solutions of lontrel with the

The synthesis of ε-NADН for the study of complexation with pesticides was carried out according to (Lichina et al., 1978; Lichina et al., 1979). Ethenonicotinamide adenine dinucleo‐

N

corresponding divalent metal salts (Aliev et al., 1988; Saratovskikh, 1989).

CH2 O P

O

OH

O P O

Methane-oxidizing bacteria *M. capsulatus* (strain M) were grown in a 20-L flow-type fermenter in a 10-L salt medium at 42°C. The rate of supply of air mixed with the gas-main natural gas

The cell suspension was collected, concentrated by separation, and washed twice with a 2.0‧ 10–2 *M* phosphate buffer, pH 7.0. The cells were destroyed in a DKM-5 semiatomated disinte‐ grator (produced at the Institute of Problems of Chemical Physics of the RAS, Chernogolovka). The cell-free preparation was centrifuged for 30 min at 3000g, the supernatant was centrifuged for 1 h at 65000g, and the precipitated membrane structures and the supernatant fraction

(SF65-1) were collected separately, frozen, and stored in liquid nitrogen until used.

OH

O H2C

N

C

**2.2. Compound synthesis**

*2.2.2. The synthesis of ε-NADН*

tide (ε-NADH) has the structure

N

N

**2.3. Enzyme inhibition**

*2.3.1. Bacterial culture preparation*

N N

N

H H HOHO

H O H

was 300+100 L min–1 (Burbaev et al., 1990). The flow rate was 0.24 m<sup>3</sup>

The degree of modification of adenine ε-NADH is 100%.

*2.2.1. The metal complexes of lontrel (ML2)*

N

N

N

H H

N

CONH2

h–1.

O

] 2Cl -

http://dx.doi.org/10.5772/54679

135

N

N

N

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

It is well known that nucleotides play an important role in organisms: energy and regulatory processes and biosynthesis. Nicotinamide adenine dinucleotide functions together with several vitally important enzymes. Therefore, it was of interest to perform a kinetic study of some widely used pesticides on the activity of the enzyme acting together with NADH of one of the enzymes performing oxidation and on nucleotide NADH.

Here we present the data showing the formation of complexes of a series of pesticides with dinucleotide NADH and data on the kinetics of NADH-OR inhibition by commercial herbi‐ cides and fungicides of various structures and several complexes of the herbicide lontrel with doubly charged metal ions.

### **2. Materials and methods**

### **2.1. Compounds, concentrations and replicates**

### *2.1.1. Pesticides*

The active substances of the herbicides and fungicides (their formulas are shown below) were isolated from commercial preparations by extraction (Saratovskikh et al., 1988). After isolation the purification was as follows: glyphosate (roundup), tachigaren, and basagran were purified by double recrystallization from water (Mel`nikov, 1987); kusagard and setoxidim were subjected to chromatography on a column with SiO2; tilt was obtained as nitrate followed by the isolation of the base; lontrel was recrystallized from benzene and then twice recrystallized from hexane; zenkor was recrystallized from hexane and then from a hexane–benzene mixture. The chemical and structural formulas of the used pesticides and lontrel metal complexes are presented in Table 2.

### *2.1.2. Metal salts*

Со, Мn, Ni, and Cu acetates and Fe lactate for the synthesis of metal complexes (pure grade, Reakhim, USSR) were purified by double recrystallization from water.

### *2.1.3. Reagents*

Commercial NADH (nicotinamide adenine dinucleotide) and NT (Sigma no. 2251, Reanal, Hungary) as an artificial electron acceptor were used.

#### **2.2. Compound synthesis**

from NADH to FAD and then to the iron sulfur 2Fe-2S cluster and to the electron acceptor. Neotetrazolium chloride (NT) was used in this work as the artificial electron acceptor.

Enzymes of this type are present in the cells of almost all organisms. Therefore, the general features of the interaction of this enzyme with pesticides can also be applied to NADH-OR

It is well known that nucleotides play an important role in organisms: energy and regulatory processes and biosynthesis. Nicotinamide adenine dinucleotide functions together with several vitally important enzymes. Therefore, it was of interest to perform a kinetic study of some widely used pesticides on the activity of the enzyme acting together with NADH of one

Here we present the data showing the formation of complexes of a series of pesticides with dinucleotide NADH and data on the kinetics of NADH-OR inhibition by commercial herbi‐ cides and fungicides of various structures and several complexes of the herbicide lontrel with

The active substances of the herbicides and fungicides (their formulas are shown below) were isolated from commercial preparations by extraction (Saratovskikh et al., 1988). After isolation the purification was as follows: glyphosate (roundup), tachigaren, and basagran were purified by double recrystallization from water (Mel`nikov, 1987); kusagard and setoxidim were subjected to chromatography on a column with SiO2; tilt was obtained as nitrate followed by the isolation of the base; lontrel was recrystallized from benzene and then twice recrystallized from hexane; zenkor was recrystallized from hexane and then from a hexane–benzene mixture. The chemical and structural formulas of the used pesticides and lontrel metal complexes are

Со, Мn, Ni, and Cu acetates and Fe lactate for the synthesis of metal complexes (pure grade,

Commercial NADH (nicotinamide adenine dinucleotide) and NT (Sigma no. 2251, Reanal,

Reakhim, USSR) were purified by double recrystallization from water.

Hungary) as an artificial electron acceptor were used.

of the enzymes performing oxidation and on nucleotide NADH.

from other organisms.

134 Herbicides - Advances in Research

doubly charged metal ions.

*2.1.1. Pesticides*

presented in Table 2.

*2.1.2. Metal salts*

*2.1.3. Reagents*

**2. Materials and methods**

**2.1. Compounds, concentrations and replicates**

### *2.2.1. The metal complexes of lontrel (ML2)*

ML2 where L is lontrel, were synthesized by refluxing ethanolic solutions of lontrel with the corresponding divalent metal salts (Aliev et al., 1988; Saratovskikh, 1989).

### *2.2.2. The synthesis of ε-NADН*

The synthesis of ε-NADН for the study of complexation with pesticides was carried out according to (Lichina et al., 1978; Lichina et al., 1979). Ethenonicotinamide adenine dinucleo‐ tide (ε-NADH) has the structure

The degree of modification of adenine ε-NADH is 100%.

### **2.3. Enzyme inhibition**

### *2.3.1. Bacterial culture preparation*

Methane-oxidizing bacteria *M. capsulatus* (strain M) were grown in a 20-L flow-type fermenter in a 10-L salt medium at 42°C. The rate of supply of air mixed with the gas-main natural gas was 300+100 L min–1 (Burbaev et al., 1990). The flow rate was 0.24 m<sup>3</sup> h–1.

The cell suspension was collected, concentrated by separation, and washed twice with a 2.0‧ 10–2 *M* phosphate buffer, pH 7.0. The cells were destroyed in a DKM-5 semiatomated disinte‐ grator (produced at the Institute of Problems of Chemical Physics of the RAS, Chernogolovka). The cell-free preparation was centrifuged for 30 min at 3000g, the supernatant was centrifuged for 1 h at 65000g, and the precipitated membrane structures and the supernatant fraction (SF65-1) were collected separately, frozen, and stored in liquid nitrogen until used.


*2.3.2. NADH-OR isolation and purification*

frozen, and stored in liquid nitrogen until used.

tion and variable NT concentrations.

*Ki* = (*I*<sup>50</sup> ⋅ *Km*) /(*SV* / *v* - *Km*),

<sup>1</sup> = (1 / *v*) / (1 / *S*) ⋅*V*

1969)

*Km*

The *K*<sup>m</sup> 1

Bowden, 1976)

*2.3.3. Determining Enzyme activity and inhibition constant (Ki*

The fraction SF65-1 (500 mL, 60 mg of protein mL–1) was passed through a column (30x7 cm) with DEAE-cellulose 52 (Whatman, UK), and the column was washed with 1 L of 2.0‧10–2 *M* phosphate buffer, pH 7.0. NADH-OR was eluted using a linear gradient of 0-0.35 *M* NaCl in the same phosphate buffer. The protein fraction with the maximum NADH-OR-activity was eluted with 0.2 *M* NaCl. The eluate was collected, concentrated under argon by ultrafiltration through the Vladipor porous membranes under a 5 atm pressure to 60 mg mL–1 of the protein, and fractionated successively on a column with Sephadex G-75 (4x70 cm) and a column with Sepharose 2B (4x80 cm) (Pharmacia, Sweden) in a 2.0‧10–2 *M* phosphate buffer, рH 7.0. The enzyme preparation with a specific activity (with respect to NT) of 1.3 µmol L–1 min–1 (mg protein)–1 (20 °C) was collected, concentrated by ultrafiltration to 21 mg mL–1 of the protein,

The activity of NADH-OR was determined from the rate of reduction of NT to formazan in a 2.0‧10–2 *M* phosphate buffer, рH 8.0. The rate of formazan formation was estimated (Burbaev et al., 1990) from the change in the absorbance at 550 nm using a Specord M-40 (GDR) spectrophotometer. The reaction was carried out in 3-mL cells (10x10 mm). The reaction mixture contained 0.1 mL of NADH-OR (1 mg of the protein), 0.3 mL of the test compound, 0.1 mL of NADH (1.0‧10–3 mol L–1), and 2.0‧10–2 *M* phosphate buffer, рH 8.0, added up to 3 mL.

The study was carried out by the traditional Michaelis–Menten procedure. The first task was to elucidate the dependence of the rate constant for the enzymatic formation of formazan on the pesticide concentration and to determine *I*50, *i.e*., the concentration of the pesticide inhibitor, resulting in a twofold decrease in the maximum rate of the enzymatic reaction. The second stage included two series of experiments: (1) at a constant NT concentration and variable NADH concentrations, and (2) at a constant NADH concentra‐

The *Ki* values were calculated from the equation (Dixon & Webb, 1979; Emanuel & Knorre,

where *Ki* is the inhibition constant; *I*<sup>50</sup> is the concentration of the pesticide inhibitor; *K*m is the determined Michaelis constant for NT or NADH; *v* is the rate; *S* is the concentration of NT or

The Hill coefficients were determined by the Hill formula (Dixon & Webb, 1979; Cornish–

1

) is the following:

NADH; *V* is the maximum rate determined from the Lineweaver–Burk plot.

The Michaelis constant in the presence of the inhibitor (*K*<sup>m</sup>

and *V* values are dictated by the inhibition type.

The reaction was initiated by adding 0.2 mL of a solution of NT (1.5‧10–3 mol L–1).

*)*

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

http://dx.doi.org/10.5772/54679

137

**Table 2.** Industrial and nomenclature names and chemical formula of the investigated substances and the complexation constants of pesticides with ε-NADH

### *2.3.2. NADH-OR isolation and purification*

**№ Common names of pesticides Chemical formula** *K,* **10-3 M**

3,6-Dichloropicolinic acid <sup>N</sup>

methylthio-1,2,4-triazin-5-one <sup>N</sup> <sup>N</sup>

1. Lontrel, Clopyralid

136 Herbicides - Advances in Research

2. Zenkor, Metribuzin

3. Basagran, Bentazon

4. Roundup, Glyphosate

5. Kusagard

6. Setoxydim

3-isopropyl-1*H*-2,1,3-

4-amino-6-tert-butyl-4,5-dihydro-3-

benzothiadiazin-4(3*H*)-one 2,2-dioxide

N-(phosphonomethyl) glycine

2-[1-(ethoxyimino)butyl]-5-[2- (ethylthio)propyl]-3-hydroxy-2-

cyclohexen-1-one

5-methyl-1,2-oxazol-3-ol

9. Lontrel metal complexes M(L)2:

complexation constants of pesticides with ε-NADH

7. Tachigaren, Hymexazol

8. Titl, Propiconazole

Sodium salt of 2-(1-allyl-oxyamino butylidene)-5,5-dimethyl-4-

methoxycarbonylcyclohexane-1,3-dione

1-[ [2-(2,4-dichlorophenyl)-4-propyl-1,3 dioxolan-2-yl]methyl]-1,2,4-triazole

**with ε-NADH**

11.7 ± 0.4

22.0 ± 2.0

2.5 ± 0.1

2.8 ± 0.7

1.8 ± 0.4

0.46 ± 0.06

O not determined

Cl Cl COOH

> NNH2 O

NCH(CH3 ) 2

(OH)2POCH2NHCH2COOH 2.2 ± 0.4

CH2 CH2 CH3

C OH H7 3C <sup>O</sup> NOC2H5

O OH N

Cl <sup>O</sup> <sup>O</sup>

N C O

Cl

C3H7

CL

CH2 CH CH2

SO <sup>N</sup> <sup>2</sup> H

N O H

Na<sup>+</sup>

(CH3)3C

\_ O

CH3

O

HC 3 HCHC 2 C2H5S

<sup>H</sup> C3

N N NCH2

> N C O

CL

Cu(L) CL <sup>2</sup> 4.6 ± 0.2 Co(L)2 3.1 ± 0.1 Ni(L)2 4.7 ± 0.3 Fe(L)2 0.55 ± 0.06 Mo(L)2 2.2 ± 0.1

**Table 2.** Industrial and nomenclature names and chemical formula of the investigated substances and the

Met O

O

CL

C

O

H3 C

> H3C C O

SCH3

The fraction SF65-1 (500 mL, 60 mg of protein mL–1) was passed through a column (30x7 cm) with DEAE-cellulose 52 (Whatman, UK), and the column was washed with 1 L of 2.0‧10–2 *M* phosphate buffer, pH 7.0. NADH-OR was eluted using a linear gradient of 0-0.35 *M* NaCl in the same phosphate buffer. The protein fraction with the maximum NADH-OR-activity was eluted with 0.2 *M* NaCl. The eluate was collected, concentrated under argon by ultrafiltration through the Vladipor porous membranes under a 5 atm pressure to 60 mg mL–1 of the protein, and fractionated successively on a column with Sephadex G-75 (4x70 cm) and a column with Sepharose 2B (4x80 cm) (Pharmacia, Sweden) in a 2.0‧10–2 *M* phosphate buffer, рH 7.0. The enzyme preparation with a specific activity (with respect to NT) of 1.3 µmol L–1 min–1 (mg protein)–1 (20 °C) was collected, concentrated by ultrafiltration to 21 mg mL–1 of the protein, frozen, and stored in liquid nitrogen until used.

#### *2.3.3. Determining Enzyme activity and inhibition constant (Ki )*

The activity of NADH-OR was determined from the rate of reduction of NT to formazan in a 2.0‧10–2 *M* phosphate buffer, рH 8.0. The rate of formazan formation was estimated (Burbaev et al., 1990) from the change in the absorbance at 550 nm using a Specord M-40 (GDR) spectrophotometer. The reaction was carried out in 3-mL cells (10x10 mm). The reaction mixture contained 0.1 mL of NADH-OR (1 mg of the protein), 0.3 mL of the test compound, 0.1 mL of NADH (1.0‧10–3 mol L–1), and 2.0‧10–2 *M* phosphate buffer, рH 8.0, added up to 3 mL. The reaction was initiated by adding 0.2 mL of a solution of NT (1.5‧10–3 mol L–1).

The study was carried out by the traditional Michaelis–Menten procedure. The first task was to elucidate the dependence of the rate constant for the enzymatic formation of formazan on the pesticide concentration and to determine *I*50, *i.e*., the concentration of the pesticide inhibitor, resulting in a twofold decrease in the maximum rate of the enzymatic reaction. The second stage included two series of experiments: (1) at a constant NT concentration and variable NADH concentrations, and (2) at a constant NADH concentra‐ tion and variable NT concentrations.

The *Ki* values were calculated from the equation (Dixon & Webb, 1979; Emanuel & Knorre, 1969)

*Ki* = (*I*<sup>50</sup> ⋅ *Km*) /(*SV* / *v* - *Km*),

where *Ki* is the inhibition constant; *I*<sup>50</sup> is the concentration of the pesticide inhibitor; *K*m is the determined Michaelis constant for NT or NADH; *v* is the rate; *S* is the concentration of NT or NADH; *V* is the maximum rate determined from the Lineweaver–Burk plot.

The Michaelis constant in the presence of the inhibitor (*K*<sup>m</sup> 1 ) is the following:

*Km* <sup>1</sup> = (1 / *v*) / (1 / *S*) ⋅*V*

The *K*<sup>m</sup> 1 and *V* values are dictated by the inhibition type.

The Hill coefficients were determined by the Hill formula (Dixon & Webb, 1979; Cornish– Bowden, 1976)

*<sup>Y</sup>* =(*Kh <sup>I</sup> <sup>h</sup>* ) /(1 + *Kh <sup>I</sup> <sup>h</sup>* ),

where *Y* is the degree of protein saturation with the ligand and is equal to the ratio of the number of occupied binding sites to the total number of binding sites; *Kh* is the association constant in the case where the concentration of the complex is as follows:

length for ε-NADH is 312 nm, and the fluorescence emission maximum is 420 nm. The fluorescence intensity of ε- NADH was measured in a 0.025 М tris-HCI buffer (рН = 6.8) at

The complexation constants of pesticides with nucleotides were calculated from the experi‐ mental titration curve for each point. The obtained values were averaged. Theoretical titration curves were calculated from the values of complexation constants obtained by the experimen‐

ESR spectra were recorded at 77 K on an SE/X2544 Radiopan radiospectrometer (Poland) at a 10 mW microwave radiation and a magnetic field modulation of 0.4 mT. The samples were prepared in a 2.0‧10–2 *M* tris-HCl buffer, рH 7.0. ESR spectra were recorded in 50% glycerol.

The study of the inhibition of NADH-OR by pesticides and metal complexes of herbicide lontrel (see Table 2) was started from the consideration of their interaction with coenzyme

It is known (Blagoyi et al., 1991) that polynucleotides, particularly, pyridinenucleotides, form complexes of various types, including charge-transfer complexes, and are highly reactive towards a series of metals. However, the introduction of the etheno group does not almost change the electronic structure of the nucleotide fragment of a NADH molecule. Therefore, the complexation of pesticides with NADH was judged about on the basis of the value of fluorescence quenching of its chemical analog, modified dinucleotide ε-NADH in which the adenine fragment is subjected to etheno-modification. Figure 1 illustrates the excitation and

С. The concentration of ε-NADH equal to 1‧10-4 М was used in experiments. The fluores‐ cence spectrum of ε-NADH was accepted to be 1, and then a solution of nucleotide was titrated in the cell with an aqueous solution of the studied pesticide in the concentration from 1‧10-8 to

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

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139

200

1‧10-2 М.

NADH.

tal data (Saratovskikh et al., 1988).

*2.4.2. Electron spin resonance analyses*

**3. Results and discussion**

**Figure 1.** Excitation and fluorescence spectra of ε-NADH.

fluorescence spectra of ε-NADH.

*Eh* <sup>⋅</sup> *Ih* <sup>=</sup> *Kh Eh* <sup>⋅</sup> *<sup>I</sup> <sup>h</sup>*

where *h* is the Hill coefficient describing the degree of allostericity and equal to the number of molecules of the ligand, in this case, the pesticide inhibitor; *I* is the concentration of the pesticide inhibitor.

### *2.3.4. Studies on bacteria Beneckea harveyi*

Lyophilized preparation of marin "luminescent" bacteria *Benechea harveyi* (strain В 1 7 – 667F) and *Photobacterium phosphoreum* (Zhmur & Orlova, 2007; Kuz`mich et al., 2002) was stored in a freezing box and used prior to use. The lyophilic preparation of bacteria was suspended in a 0.85% solution of NaCl.

To determine toxicity, 0.3–0.5 mL of suspended bacteria was added to 0.5 mL of the studied water. A 0.85% solution of NaCl or water from an aquarium was used as a control. The measurements were carried out by the instrumental method with a BLM-8801 luminometer (SKTB "Nauka," USSR) with detection on a voltmeter by a decrease in the bioluminescence intensity in the presence of a sample of analyzed water compared to the control.

The 50% (and more) decrease in the luminescence intensity indicates that the aqueous medium is toxic. The bioluminescence intensity of bacteria is determined by the activity of intracellular metabolic processes involving the luciferase enzyme. The decrease in luminescence can be due to the inhibition of the enzyme itself and to the influence of toxicants to other units of the metabolic chain.

The toxicity coefficient was calculated by the formula

#### Т = (Ic- It ) / It ×100%,

where Ic is the bioluminescence intensity in the control, and It is the luminescence intensity in the tested sample.

At Т ≤ 19% the tested sample is not toxic. At 19 < Т ≤ 50% the tested sample is considered toxic, whereas at T > 50% the sample is strongly toxic.

Each toxicological experiment was carried out at least three times and then the results obtained were statistically processed.

### **2.4. Instrumental analysis**

### *2.4.1. Fluorimetric measurements*

The fluorescence spectra of the etheno-modified compounds were recorded on an Aminco– Bowman spectrofluorimenter (US) in 3.5-mL quartz cells. The fluorescence excitation wave‐ length for ε-NADH is 312 nm, and the fluorescence emission maximum is 420 nm. The fluorescence intensity of ε- NADH was measured in a 0.025 М tris-HCI buffer (рН = 6.8) at 200 С. The concentration of ε-NADH equal to 1‧10-4 М was used in experiments. The fluores‐ cence spectrum of ε-NADH was accepted to be 1, and then a solution of nucleotide was titrated in the cell with an aqueous solution of the studied pesticide in the concentration from 1‧10-8 to 1‧10-2 М.

The complexation constants of pesticides with nucleotides were calculated from the experi‐ mental titration curve for each point. The obtained values were averaged. Theoretical titration curves were calculated from the values of complexation constants obtained by the experimen‐ tal data (Saratovskikh et al., 1988).

### *2.4.2. Electron spin resonance analyses*

*<sup>Y</sup>* =(*Kh <sup>I</sup> <sup>h</sup>* ) /(1 + *Kh <sup>I</sup> <sup>h</sup>* ),

138 Herbicides - Advances in Research

*Eh* <sup>⋅</sup> *Ih* <sup>=</sup> *Kh Eh* <sup>⋅</sup> *<sup>I</sup> <sup>h</sup>*

a 0.85% solution of NaCl.

metabolic chain.

the tested sample.

) / It ×100%,

were statistically processed.

**2.4. Instrumental analysis**

*2.4.1. Fluorimetric measurements*

Т = (Ic- It

*2.3.4. Studies on bacteria Beneckea harveyi*

The toxicity coefficient was calculated by the formula

whereas at T > 50% the sample is strongly toxic.

inhibitor.

where *Y* is the degree of protein saturation with the ligand and is equal to the ratio of the number of occupied binding sites to the total number of binding sites; *Kh* is the association

where *h* is the Hill coefficient describing the degree of allostericity and equal to the number of molecules of the ligand, in this case, the pesticide inhibitor; *I* is the concentration of the pesticide

Lyophilized preparation of marin "luminescent" bacteria *Benechea harveyi* (strain В 1 7 – 667F) and *Photobacterium phosphoreum* (Zhmur & Orlova, 2007; Kuz`mich et al., 2002) was stored in a freezing box and used prior to use. The lyophilic preparation of bacteria was suspended in

To determine toxicity, 0.3–0.5 mL of suspended bacteria was added to 0.5 mL of the studied water. A 0.85% solution of NaCl or water from an aquarium was used as a control. The measurements were carried out by the instrumental method with a BLM-8801 luminometer (SKTB "Nauka," USSR) with detection on a voltmeter by a decrease in the bioluminescence

The 50% (and more) decrease in the luminescence intensity indicates that the aqueous medium is toxic. The bioluminescence intensity of bacteria is determined by the activity of intracellular metabolic processes involving the luciferase enzyme. The decrease in luminescence can be due to the inhibition of the enzyme itself and to the influence of toxicants to other units of the

where Ic is the bioluminescence intensity in the control, and It is the luminescence intensity in

At Т ≤ 19% the tested sample is not toxic. At 19 < Т ≤ 50% the tested sample is considered toxic,

Each toxicological experiment was carried out at least three times and then the results obtained

The fluorescence spectra of the etheno-modified compounds were recorded on an Aminco– Bowman spectrofluorimenter (US) in 3.5-mL quartz cells. The fluorescence excitation wave‐

intensity in the presence of a sample of analyzed water compared to the control.

constant in the case where the concentration of the complex is as follows:

ESR spectra were recorded at 77 K on an SE/X2544 Radiopan radiospectrometer (Poland) at a 10 mW microwave radiation and a magnetic field modulation of 0.4 mT. The samples were prepared in a 2.0‧10–2 *M* tris-HCl buffer, рH 7.0. ESR spectra were recorded in 50% glycerol.

### **3. Results and discussion**

The study of the inhibition of NADH-OR by pesticides and metal complexes of herbicide lontrel (see Table 2) was started from the consideration of their interaction with coenzyme NADH.

**Figure 1.** Excitation and fluorescence spectra of ε-NADH.

It is known (Blagoyi et al., 1991) that polynucleotides, particularly, pyridinenucleotides, form complexes of various types, including charge-transfer complexes, and are highly reactive towards a series of metals. However, the introduction of the etheno group does not almost change the electronic structure of the nucleotide fragment of a NADH molecule. Therefore, the complexation of pesticides with NADH was judged about on the basis of the value of fluorescence quenching of its chemical analog, modified dinucleotide ε-NADH in which the adenine fragment is subjected to etheno-modification. Figure 1 illustrates the excitation and fluorescence spectra of ε-NADH.

NH

C O

+

Cl

N

It is known (Blagoyi et al., 1991) that polynucleotides, particularly, pyridinenucleotides, form complexes of various types, including

quenching proceeds via the Stern–Volmer mechanism due to random collisions. Therefore, the result of quenching is the formation

Figure 3. Dependences of the fluorescence intensity of ε-NADH on the concentration of the lontrel metal complexes: (1) Fe(L)2; (2) Ni(L)2; (3)

The mathematical model of the process was considered to refine the mechanism of formation of complexes [ε-NADH–pesticide] and to estimate their stability constants. It was assumed that the pesticides interact with ε-NADH according to the scheme

where А is the concentration of etheno-modified units of the nucleotide (adenine) in ε-NADH, Р is the pesticide concentration, П is

Equations 2–4 make it possible to determine the values of the complexation constant from the experimentally determined

where Io is the fluorescence intensity of free ε-NADH, Iк is the limiting value of fluorescence intensity of ε-NADH at the maximum

The value of stoichiometric coefficient n should preliminarily be determined from the data obtained at a rather high pesticide

concentration of the quencher, and I is the fluorescence intensity of ε-NADH at the given concentration of the quencher.

*<sup>A</sup>* { *<sup>P</sup>*<sup>0</sup> <sup>−</sup>*n*( *<sup>A</sup>*<sup>0</sup> <sup>−</sup> *<sup>A</sup>* )}*<sup>n</sup>* (5)

is the complexation constant, and n is the stoichiometric coefficient

1 1 

*<sup>к</sup>* <sup>−</sup><sup>1</sup> <sup>→</sup> *<sup>П</sup>* (1)

*K A* × *P <sup>n</sup>* = *П* (2)

*А* + *П* = *А*<sup>0</sup> (3) *P* + *n П* = *P*<sup>0</sup> (4)

Cu(L)2. The concentration of ε-NADH is 1∙10-5 М. Solid lines are theoretical curves, and points are experimental data.

**3**

**consentration, Po x10-4, M**

**1**

**2**

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

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141

1 <sup>1</sup> A + n P <sup>П</sup> *<sup>к</sup> к* (1)

0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1

are experimental data.

*K* =*κ* +1 / *κ* <sup>−</sup><sup>1</sup>

*<sup>A</sup>* <sup>=</sup> *<sup>I</sup>* <sup>−</sup> *<sup>I</sup><sup>κ</sup> I*<sup>0</sup> − *I<sup>κ</sup>*

*A*0,

number of equivalent binding sites.

**Fluorescense, I/Io**

<sup>n</sup> K A × P = <sup>П</sup> (2)

А <sup>о</sup> + П = [ ] А (3)

<sup>o</sup> P + ] n П =P[ (4)

concentration

experimentally determined concentration

A = A, <sup>o</sup> *I I I I* 

o o [] ( A P >>n A – [ ]). (6)

Inline formula

[ ][] [ ]{[ ] ([ ] [ ])} K = *<sup>n</sup> A A A P nA A* 

intensity of ε-NADH at the given concentration of the quencher.

 

(5)

*<sup>K</sup>* <sup>=</sup> *<sup>A</sup>*<sup>0</sup> <sup>−</sup> *<sup>A</sup>*

concentration at which the following equation is fulfilled:

After condition (6) is fulfilled, equation (5) can be rewritten in the form

 

the concentration of the reaction product = complex, K =

0,0 1,0 2,0 3,0 4,0 5,0

**Figure 3.** Dependences of the fluorescence intensity of ε-NADH on the concentration of the lontrel metal complexes: (1) Fe(L)2; (2) Ni(L)2; (3) Cu(L)2. The concentration of ε-NADH is 1‧10-5 М. Solid lines are theoretical curves, and points

where А is the concentration of etheno-modified units of the nucleotide (adenine) in ε-NADH, Р is the pesticide concentration, П is the concentration of the reaction product = complex,

Equations 2–4 make it possible to determine the values of the complexation constant from the

where Io is the fluorescence intensity of free ε-NADH, Iк is the limiting value of fluorescence intensity of ε-NADH at the maximum concentration of the quencher, and I is the fluorescence

At equilibrium the process is described by the following system of equations:

is the complexation constant, and n is the stoichiometric coefficient equal to the

equal to the number of equivalent binding sites.

At equilibrium the process is described by the following system of equations:

*<sup>A</sup>* <sup>+</sup> *<sup>n</sup> <sup>P</sup>* <sup>←</sup> *<sup>к</sup>* +1

Figure 2. Dependences of the fluorescence intensity of ε-NADH on the pesticide concentration: (1) tilt; (2) kusagard; (3) zenkor. The concentration

of a covalent bond with the adenine fragment, as it is shown in the scheme of the [NADH–lontrel] complex

Figures 2 and 3 represent the obtained dependences of the change in the fluorescence intensity of ε-NADH on the concentration of various quenchers. When the concentration of pesticide (or metal complex) increases, the fluorescence quenching of compound ε-NADH is observed, which is not accompanied by a shift of the position of the excitation maximum and fluorescence emission. The absence of spectral changes in all cases considered indicates the absence of changes in the ground and excited levels of the modified based upon the interaction with pesticides. Fluorescence quenching was observed at the pesticide and lontrel metal complexes concentrations ranging from 10-6 to 10-3 М. Such low concentrations of the quencher exclude the assumption that the quenching proceeds via the Stern–Volmer mechanism due to random collisions. Therefore, the result of quenching is the formation of a covalent bond with the adenine fragment, as it is shown in the scheme of the [NADH–lontrel] complex Scheme 3. Add caption Figures 2 and 3 represent the obtained dependences of the change in the fluorescence intensity of ε-NADH on the concentration of various quenchers. When the concentration of pesticide (or metal complex) increases, the fluorescence quenching of compound ε-NADH is observed, which is not accompanied by a shift of the position of the excitation maximum and fluorescence emission. The absence of spectral changes in all cases considered indicates the absence of changes in the ground and excited levels of the modified based upon the interaction with pesticides. Fluorescence quenching was observed at the pesticide and lontrel metal complexes concentrations ranging from 10-6 to 10-3 М. Such low concentrations of the quencher exclude the assumption that the N N N N R

of ε-NADH is 1∙10-5 М. Solid lines are theoretical curves, and points are experimental data. **Figure 2.** Dependences of the fluorescence intensity of ε-NADH on the pesticide concentration: (1) tilt; (2) kusagard; (3) zenkor. The concentration of ε-NADH is 1‧10-5 М. Solid lines are theoretical curves, and points are experimental data.

The mathematical model of the process was considered to refine the mechanism of formation of complexes [ε-NADH–pesticide] and to estimate their stability constants. It was assumed that the pesticides interact with ε-NADH according to the scheme

Figures 2 and 3 represent the obtained dependences of the change in the fluorescence intensity of ε-NADH on the concentration of various quenchers. When the concentration of pesticide (or metal complex) increases, the fluorescence quenching of compound ε-NADH is observed, which is not accompanied by a shift of the position of the excitation maximum and fluorescence emission. The Cu(L)2. The concentration of ε-NADH is 1∙10-5 М. Solid lines are theoretical curves, and points are experimental data. The mathematical model of the process was considered to refine the mechanism of formation of complexes [ε-NADH–pesticide] **Figure 3.** Dependences of the fluorescence intensity of ε-NADH on the concentration of the lontrel metal complexes: (1) Fe(L)2; (2) Ni(L)2; (3) Cu(L)2. The concentration of ε-NADH is 1‧10-5 М. Solid lines are theoretical curves, and points are experimental data.

$$\mathbb{L}[A] + n[P] \leftarrow \frac{\kappa^{\*1}}{\kappa^{-1}} - \mathbb{L}[II] \tag{1}$$

Figure 3. Dependences of the fluorescence intensity of ε-NADH on the concentration of the lontrel metal complexes: (1) Fe(L)2; (2) Ni(L)2; (3)

*к* where А is the concentration of etheno-modified units of the nucleotide (adenine) in ε-NADH, Р is the pesticide concentration, П is the concentration of the reaction product = complex, K = 1 1 is the complexation constant, and n is the stoichiometric coefficient equal to the number of equivalent binding sites. where А is the concentration of etheno-modified units of the nucleotide (adenine) in ε-NADH, Р is the pesticide concentration, П is the concentration of the reaction product = complex, *K* =*κ* +1 / *κ* <sup>−</sup><sup>1</sup> is the complexation constant, and n is the stoichiometric coefficient equal to the number of equivalent binding sites.

At equilibrium the process is described by the following system of equations: At equilibrium the process is described by the following system of equations:

concentration

A = A, <sup>o</sup> *I I*

o o [] ( A P >>n A – [ ]). (6)

$$K \ulcorner A \urcorner \ltimes \ulcorner P \urcorner \ulcorner^\* \urcorner = \ulcorner \ulcorner T \urcorner \urcorner \tag{2}$$

$$\begin{array}{c} \stackrel{\cdot}{\mathbf{L}} \stackrel{\cdot}{\mathbf{J}} + \stackrel{\cdot}{\mathbf{L}} \stackrel{\cdot}{\mathbf{J}} = \stackrel{\cdot}{\mathbf{L}} A\_0 \end{array} \tag{3}$$

$$\begin{bmatrix} P \end{bmatrix} + n \begin{bmatrix} IT \end{bmatrix} = \begin{bmatrix} P\_0 \end{bmatrix} \tag{4}$$

Equations 2–4 make it possible to determine the values of the complexation constant from the experimentally determined

The value of stoichiometric coefficient n should preliminarily be determined from the data obtained at a rather high pesticide

<sup>o</sup> P + ] n П =P[ (4) Equations 2–4 make it possible to determine the values of the complexation constant from the experimentally determined concentration

$$\mathbf{L} \mathbf{A} \mathbf{J} = \frac{I - I\_{\kappa}}{I\_0 - I\_{\kappa}} A\_0 \mathbf{J}$$

N

Cl

Cl

It is known (Blagoyi et al., 1991) that polynucleotides, particularly, pyridinenucleotides, form complexes of various types, including charge-transfer complexes, and are highly reactive towards a series of metals. However, the introduction of the etheno group does not almost change the electronic structure of the nucleotide fragment of a NADH molecule. Therefore, the complexation of pesticides with NADH was judged about on the basis of the value of fluorescence quenching of its chemical analog, modified dinucleotide ε-NADH in which the adenine fragment is subjected to etheno-modification. Figure 1 illustrates the excitation and

absence of spectral changes in all cases considered indicates the absence of changes in the ground and excited levels of the modified based upon the interaction with pesticides. Fluorescence quenching was observed at the pesticide and lontrel metal complexes concentrations ranging from 10-6 to 10-3 М. Such low concentrations of the quencher exclude the assumption that the quenching proceeds via the Stern–Volmer mechanism due to random collisions. Therefore, the result of quenching is the formation

of a covalent bond with the adenine fragment, as it is shown in the scheme of the [NADH–lontrel] complex

**1**

of ε-NADH is 1∙10-5 М. Solid lines are theoretical curves, and points are experimental data.

**concentration, P0**

x10-3 , M

0,0 0,5 1,0 1,5 2,0

**Figure 2.** Dependences of the fluorescence intensity of ε-NADH on the pesticide concentration: (1) tilt; (2) kusagard; (3) zenkor. The concentration of ε-NADH is 1‧10-5 М. Solid lines are theoretical curves, and points are experimental

The mathematical model of the process was considered to refine the mechanism of formation of complexes [ε-NADH–pesticide] and to estimate their stability constants. It was assumed

**2**

N

+

N

C O

Figure 1. Excitation and fluorescence spectra of ε-NADH.

R

Figures 2 and 3 represent the obtained dependences of the change in the fluorescence intensity of ε-NADH on the concentration of various quenchers. When the concentration of pesticide (or metal complex) increases, the fluorescence quenching of compound ε-NADH is observed, which is not accompanied by a shift of the position of the excitation maximum and fluorescence emission. The absence of spectral changes in all cases considered indicates the absence of changes in the ground and excited levels of the modified based upon the interaction with pesticides. Fluorescence quenching was observed at the pesticide and lontrel metal complexes concentrations ranging from 10-6 to 10-3 М. Such low concentrations of the quencher exclude the assumption that the quenching proceeds via the Stern–Volmer mechanism due to random collisions. Therefore, the result of quenching is the formation of a covalent bond with the

N

Cl

fluorescence spectra of ε-NADH.

N

N

+

Scheme 3. Add caption

N

C O

R

N

140 Herbicides - Advances in Research

NH

N

0,0

data.

0,2

0,4

0,6

**Fluorescence, I/Io**

0,8

1,0

H N

N

Cl

adenine fragment, as it is shown in the scheme of the [NADH–lontrel] complex

**3**

that the pesticides interact with ε-NADH according to the scheme

*I I* Inline formula where Io is the fluorescence intensity of free ε-NADH, Iк is the limiting value of fluorescence intensity of ε-NADH at the maximum concentration of the quencher, and I is the fluorescence intensity of ε-NADH at the given concentration of the quencher. where Io is the fluorescence intensity of free ε-NADH, Iк is the limiting value of fluorescence intensity of ε-NADH at the maximum concentration of the quencher, and I is the fluorescence intensity of ε-NADH at the given concentration of the quencher.

concentration at which the following equation is fulfilled:

After condition (6) is fulfilled, equation (5) can be rewritten in the form

$$K = \frac{\mathbf{[\![A\_0]\!]} \mathbf{[\![A]\!]}}{\mathbf{[\![A]\!] [\![P\_0]\!] - n(\![A\_0]\!] - \![A\!]) \mathbf{[\![A]\!]}} \tag{5}$$

The value of stoichiometric coefficient n should preliminarily be determined from the data obtained at a rather high pesticide concentration at which the following equation is fulfilled:

$$\mathbb{I}[P\_o] \succeq n \{ \mathbb{I}A\_o \mathbb{I}\text{-}\mathbb{I}A \}. \tag{6}$$

values found for n and K. Satisfactory coincidence of the experimental and theoretical data

As can be seen from Table 2, of the synthesized pesticides, zenkor has the lowest complexation

noteworthy that the complexation constant of the lontrel metal complexes with ε-NADH is substantially lower than the corresponding constant for lontrel. It is known (Luisi et al., 1975) than in solution NADH exists predominantly in a folded conformation in which the adenine part of the molecule is localized near the nicotine amide part of the nucleotide. About 90% dinucleotide exists in this conformation in solution. The rest 10% exist in solution in the "open" conformation when the nicotine amide part is remote from the adenine structure. Therefore, it can be assumed that the decrease in the complexation constants with ε-NADH for the metal complexes compared to lontrel indicates steric hindrances appeared upon the formation of the

0,0 0,2 0,4 0,6 0,8 1,0

S1 , 10<sup>3</sup> S M 1, 103 M

**Figure 5.** Kinetic curves for the oxidation rate of NADH-OR *vs*. the concentration of NADH at a constant concentration of NT in the absence of an inhibitor (1) and in the presence of 0.33‧10–4 (2) and 1.00‧10–4 mol L–1 (3) of lontrel; *C*NT =

The effect of pesticides on the activity of NADH-OR is illustrated by Figs 5–8. The experimental kinetic curves for the rate of NADH-OR oxidation *vs.* concentration of the NADH (*S*1) substrate at an invariable NT concentration are presented in Fig. 5. The plots converted to the Linewea‐ ver–Burk coordinates are shown in Figs 6–8. Figure 6 shows the pattern of OR inhibition by lontrel as a function of the concentration of NADH (at a constant NT concentration). The intersection of these straight lines in one point on the ordinate (see Fig. 6) indicates that the herbicide lontrel inhibits NADH-OR and competes with NADH for the region of binding with the enzyme. The 1/*S*1 intercept on the abscissa was used to calculate the inhibition constant

М-1) and tilt has the highest one (K = 4.6‧10<sup>2</sup>

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

**3**

**2**

**1**

М-1). It is

143

http://dx.doi.org/10.5772/54679

indicates that the developed model is valid.

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

2.467‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NADH = 1.0‧10–4 - 1.5‧10–3 mol L–1.

(*S*1 is the NADH concentration, *S*2 is the NT concentration).

. v, 106 M сек-1

v, 106

M·s-1

constant (K) with ε-NADH (K = 2.1‧10<sup>4</sup>

[NADH–M(L)2] complex.

After condition (6) is fulfilled, equation (5) can be rewritten in the form

determination will be unacceptably high.

Figure 4. Dependences of the ln ����

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

. v, 106 M сек-1

$$\ln\left(\frac{\mathbb{E}A\_0\mathbb{J}-\mathbb{E}A}{\mathbb{E}A}\right) = \ln K + n\ln\mathbb{E}P\_0\mathbb{J}\tag{7}$$

from which it follows that the dependence of ln ln *A*<sup>0</sup> − *A <sup>A</sup>* on ln[Ро] is a straight line with the angular coefficient equal to n. (7)

As can be seen from Fig. 4, stoichiometric coefficient n is equal to 1±0.2 for all pesticides and metal complexes evaluated. Therefore, we may conclude that only one pesticide molecule interact with one molecule of ε-NADH. Having determined the value of n at high Ро, one can find the value of K at other pesticide concentrations. The use values of Ро should not be too low, since at very low [Ро] relative errors of the values of ([Ао] – [A]) and ([Pо] – n[Ао] – [А]) can be too high and the errors of complexation constant determination will be unacceptably high. <sup>o</sup> ln = lnK + n ln [ ][] ( ) [] P , [ ] *A A A* from which it follows that the dependence of ln [ ][] [ ] *A A A* on ln[Ро] is a straight line with the angular coefficient equal to n. As can be seen from Fig. 4, stoichiometric coefficient n is equal to 1±0.2 for all pesticides and metal complexes evaluated. Therefore, we may conclude that only one pesticide molecule interact with one molecule of ε-NADH. Having determined the value of n at high Ро, one can find the value of K at other pesticide concentrations. The use values of Ро should not be too low, since at very low [Ро] relative errors of the values of ([Ао] – [A]) and ([Pо] – n[Ао] – [А]) can be too high and the errors of complexation constant

**Figure 4.** Dependences of the ln *Ao* - *<sup>A</sup> <sup>A</sup>* on the lnPo. (1) zenkor; (2) Cu(L)2; (3) setoxidim; (4) Mo(L)2; (5) Co(L)2.

appeared upon the formation of the [NADH–M(L)2] complex.

0,0 0,2 0,4 0,6 0,8 1,0

S1 , 10<sup>3</sup> M

Сu(lontrel)2 complexes (Сu(L)2) calculated by the values found for n and K. Satisfactory coincidence of the experimental and theoretical data indicates that the developed model is valid. Figures 2 and 3 show the experimental dependences of I/I<sup>о</sup> on [Ро] and the corresponding theoretical curves for the pesticides and Сu(lontrel)<sup>2</sup> complexes (Сu(L)2) calculated by the

� on the lnPo. (1) zenkor; (2) Cu(L)2; (3) setoxidim; (4) Mo(L)2; (5) Co(L)2.

Figures 2 and 3 show the experimental dependences of I/Iо on [Ро] and the corresponding theoretical curves for the pesticides and

As can be seen from Table 2, of the synthesized pesticides, zenkor has the lowest complexation constant (K) with ε-NADH (K = 2.1·10<sup>4</sup> М-1) and tilt has the highest one (K = 4.6·10<sup>2</sup> М-1). It is noteworthy that the complexation constant of the lontrel metal complexes with ε-NADH is substantially lower than the corresponding constant for lontrel. It is known (Luisi et al., 1975) than in solution NADH exists predominantly in a folded conformation in which the adenine part of the molecule is localized near the nicotine amide part of the nucleotide. About 90% dinucleotide exists in this conformation in solution. The rest 10% exist in solution in the "open" conformation when the nicotine amide part is remote from the adenine structure. Therefore, it can be assumed that the decrease in the complexation constants with ε-NADH for the metal complexes compared to lontrel indicates steric hindrances

**3**

Figure 5. Kinetic curves for the oxidation rate of NADH-OR *vs*. the concentration of NADH at a constant concentration

**2**

**1**

values found for n and K. Satisfactory coincidence of the experimental and theoretical data indicates that the developed model is valid.

The value of stoichiometric coefficient n should preliminarily be determined from the data obtained at a rather high pesticide concentration at which the following equation is fulfilled:

As can be seen from Fig. 4, stoichiometric coefficient n is equal to 1±0.2 for all pesticides and metal complexes evaluated. Therefore, we may conclude that only one pesticide molecule interact with one molecule of ε-NADH. Having determined the value of n at high Ро, one can find the value of K at other pesticide concentrations. The use values of Ро should not be too low, since at very low [Ро] relative errors of the values of ([Ао] – [A]) and ([Pо] – n[Ао] – [А]) can be too high and the errors of complexation constant determination will be unacceptably


**2 1**

*<sup>A</sup>* on the lnPo. (1) zenkor; (2) Cu(L)2; (3) setoxidim; (4) Mo(L)2; (5) Co(L)2.

**5**

Figures 2 and 3 show the experimental dependences of I/I<sup>о</sup> on [Ро] and the corresponding theoretical curves for the pesticides and Сu(lontrel)<sup>2</sup> complexes (Сu(L)2) calculated by the

**4**

After condition (6) is fulfilled, equation (5) can be rewritten in the form

ln( *<sup>A</sup>*<sup>0</sup> <sup>−</sup> *<sup>A</sup>*

from which it follows that the dependence of ln ln

<sup>o</sup> ln = lnK + n ln [ ][] ( ) [] P , [ ] *A A*

(7)

determination will be unacceptably high.

Figure 4. Dependences of the ln ����


**Figure 4.** Dependences of the ln *Ao* - *<sup>A</sup>*

**ln(Ao-A)/A**

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

. v, 106 M сек-1

theoretical data indicates that the developed model is valid.

appeared upon the formation of the [NADH–M(L)2] complex.

0,0 0,2 0,4 0,6 0,8 1,0

S1 , 10<sup>3</sup> M

from which it follows that the dependence of ln [ ][]

the angular coefficient equal to n.

142 Herbicides - Advances in Research

*A* 

high.

*Po* > >*n*( *Ao* − *A* ). (6)

*<sup>A</sup>* ) =ln*<sup>K</sup>* <sup>+</sup> *<sup>n</sup>*ln *<sup>P</sup>*<sup>0</sup> , (7)

*<sup>A</sup>* on ln[Ро] is a straight line with

As can be seen from Fig. 4, stoichiometric coefficient n is equal to 1±0.2 for all pesticides and metal complexes evaluated. Therefore, we may conclude that only one pesticide molecule interact with one molecule of ε-NADH. Having determined the value of n at high Ро, one can find the value of K at other pesticide concentrations. The use values of Ро should not be too low, since at very low [Ро] relative errors of the values of ([Ао] – [A]) and ([Pо] – n[Ао] – [А]) can be too high and the errors of complexation constant

� on the lnPo. (1) zenkor; (2) Cu(L)2; (3) setoxidim; (4) Mo(L)2; (5) Co(L)2.

**lnPo**

**3**


As can be seen from Table 2, of the synthesized pesticides, zenkor has the lowest complexation constant (K) with ε-NADH (K = 2.1·10<sup>4</sup> М-1) and tilt has the highest one (K = 4.6·10<sup>2</sup> М-1). It is noteworthy that the complexation constant of the lontrel metal complexes with ε-NADH is substantially lower than the corresponding constant for lontrel. It is known (Luisi et al., 1975) than in solution NADH exists predominantly in a folded conformation in which the adenine part of the molecule is localized near the nicotine amide part of the nucleotide. About 90% dinucleotide exists in this conformation in solution. The rest 10% exist in solution in the "open" conformation when the nicotine amide part is remote from the adenine structure. Therefore, it can be assumed that the decrease in the complexation constants with ε-NADH for the metal complexes compared to lontrel indicates steric hindrances

**3**

Figure 5. Kinetic curves for the oxidation rate of NADH-OR *vs*. the concentration of NADH at a constant concentration

**2**

**1**

on ln[Ро] is a straight line with the angular coefficient equal to n.

*A*<sup>0</sup> − *A*

[ ] *A A A* 

As can be seen from Table 2, of the synthesized pesticides, zenkor has the lowest complexation constant (K) with ε-NADH (K = 2.1‧10<sup>4</sup> М-1) and tilt has the highest one (K = 4.6‧10<sup>2</sup> М-1). It is noteworthy that the complexation constant of the lontrel metal complexes with ε-NADH is substantially lower than the corresponding constant for lontrel. It is known (Luisi et al., 1975) than in solution NADH exists predominantly in a folded conformation in which the adenine part of the molecule is localized near the nicotine amide part of the nucleotide. About 90% dinucleotide exists in this conformation in solution. The rest 10% exist in solution in the "open" conformation when the nicotine amide part is remote from the adenine structure. Therefore, it can be assumed that the decrease in the complexation constants with ε-NADH for the metal complexes compared to lontrel indicates steric hindrances appeared upon the formation of the [NADH–M(L)2] complex.

**Figure 5.** Kinetic curves for the oxidation rate of NADH-OR *vs*. the concentration of NADH at a constant concentration of NT in the absence of an inhibitor (1) and in the presence of 0.33‧10–4 (2) and 1.00‧10–4 mol L–1 (3) of lontrel; *C*NT = 2.467‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NADH = 1.0‧10–4 - 1.5‧10–3 mol L–1.

Figures 2 and 3 show the experimental dependences of I/Iо on [Ро] and the corresponding theoretical curves for the pesticides and Сu(lontrel)2 complexes (Сu(L)2) calculated by the values found for n and K. Satisfactory coincidence of the experimental and The effect of pesticides on the activity of NADH-OR is illustrated by Figs 5–8. The experimental kinetic curves for the rate of NADH-OR oxidation *vs.* concentration of the NADH (*S*1) substrate at an invariable NT concentration are presented in Fig. 5. The plots converted to the Linewea‐ ver–Burk coordinates are shown in Figs 6–8. Figure 6 shows the pattern of OR inhibition by lontrel as a function of the concentration of NADH (at a constant NT concentration). The intersection of these straight lines in one point on the ordinate (see Fig. 6) indicates that the herbicide lontrel inhibits NADH-OR and competes with NADH for the region of binding with the enzyme. The 1/*S*1 intercept on the abscissa was used to calculate the inhibition constant (*S*1 is the NADH concentration, *S*2 is the NT concentration).

It can be seen from Fig. 7 that the herbicide roundup does not compete with NADH for the

3

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

http://dx.doi.org/10.5772/54679

145

2

are equal

1


**Figure 7.** Inhibition of the NADH-oxidoreductase with roundup (in the Lineweaver-Burk coordinates) in the absence of the inhibitor (1) and in the presence of 1.17‧10–3 (2) and 2.50‧10–3 mol L–1 (*3*) roundup; *C*NT = 2.467‧10–3 mol L–1;

The dependences of the reciprocal reaction rate on the reciprocal concentration of the NT electron acceptor (with the NADH concentration remaining constant) are shown in Figs 8 and 9. The herbicides lontrel and zenkor (see Fig. 8, Table 3) also inhibit the rate of electron transfer

to 7.42‧10–4 and 8.94‧10–4 mol L–1, respectively). The lontrel complex with the copper ion exhibits noncompetitive inhibition, while the complex with cobalt exerts mixed inhibition (see Fig. 9,

The Michaelis constants (*K*m) calculated without inhibitors are 6.6‧10–4 and 2.47‧10–3 mol L–1 for NADH and NT, respectively. It was determined in the preliminary experiment that all

The data on the effect of other herbicides, fungicides, and lontrel metal complexes on the rate of NADH oxidation and the rate of NT reduction with NADH-oxidoreductase are presented

Of all the compounds studied, the highest inhibitory activities were found for zenkor and basagran (*I*50 are 5.0‧10–4 and 6.0‧10–4 mol L–1, respectively). Lontrel, roundup, tachigaren, and tilt inhibit NADH-OR somewhat less efficient (*I*<sup>50</sup> are 1.1‧10–3, 1.7‧10–3, 2.7‧10–3, and 2.2‧10–3 mol L–1, respectively, see Table 3). Kusagard and setoxidim exhibit weak antireductase activities; they depress the enzyme activity when are present in higher concentrations: 2.7‧10–2 and 1.7‧

from the NADH-OR active center to NT. The inhibition pattern is uncompetitive (*K*<sup>i</sup>

1/S1 , 10<sup>3</sup> <sup>M</sup>-1 1/S1, 103 M-1

0.5

1.0

1/V, 106 M-1сек

1/v, 106 M-1·s

*C*enzyme = 1.0‧10–6 mol L–1; *C*NADH = 2.0‧10–4 - 1.5‧10–3 mol L–1.

compounds under study reversibly inhibit NADH-OR.

Table 4).

in Table 3.

1.5

enzyme binding site.

**Figure 6.** Inhibition of the NADH-oxidoreductase by lontrel (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 0.33‧10–4 (2) and 1.00‧10–4 mol L–1 (3) of lontrel; *C*NT = 2.467‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NADH = 1.0‧10–4 - 1.5‧10–3 mol L–1.

It can be seen from Tables 3 and 5 that the lontrel complex with the copper ion, although follows a competitive mechanism of inhibition with respect to NADH, still inhibits the oxidation of NADH almost 30 times stronger than the parent lontrel. The *I*50 values are equal to 1.1‧10–3 and 3.3‧10–4 mol L–1 (*K*<sup>i</sup> are 1.0‧10–4 and 6‧10–6 mol L–1).


\* In the absence of an inhibitor *V*max = 7.40‧10–6 mol L–1 s–1; *S*1 = 6.58‧10–3 mol L–1; *S*2 = 2.65‧10–3 mol L–1.

\*\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

**Table 3.** Effect of inhibitors on NADH-oxidoreductase\*

It can be seen from Fig. 7 that the herbicide roundup does not compete with NADH for the enzyme binding site.

1/S1, 103 M-1

**Figure 6.** Inhibition of the NADH-oxidoreductase by lontrel (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 0.33‧10–4 (2) and 1.00‧10–4 mol L–1 (3) of lontrel; *C*NT = 2.467‧10–3 mol L–1; *C*enzyme =

It can be seen from Tables 3 and 5 that the lontrel complex with the copper ion, although follows a competitive mechanism of inhibition with respect to NADH, still inhibits the oxidation of NADH almost 30 times stronger than the parent lontrel. The *I*50 values are equal to 1.1‧10–3 and

*h* **for NADH for NT**

*S1 Ki*

\* In the absence of an inhibitor *V*max = 7.40‧10–6 mol L–1 s–1; *S*1 = 6.58‧10–3 mol L–1; *S*2 = 2.65‧10–3 mol L–1.

\*\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

**Table 3.** Effect of inhibitors on NADH-oxidoreductase\*

Zencor 5.00∙10-4 1.952 - 4.93∙10-3 0.25 A 0.23∙10-6 3.39∙10-4 8.94 B Lontrel (L) 1.10∙10-3 1.726 - 1.23∙10-3 1.00 A 1.88∙10-6 6.98∙10-4 7.42 B Bazagran 6.00∙10-4 2.086 1.82∙10-6 1.83∙10-4 12.80 B 0.26∙10-6 2.55∙10-4 8.40 B Kuzagard 27.0∙10-2 1.575 - 9.86∙10-3 14.00 A - 5.72∙10-3 158.9 A Tachigaren 2.70∙10-3 1.920 - 2.47∙10-3 21.00 A - 5.30∙10-3 4.55 A Roundup 1.70∙10-3 1.328 3.33∙10-6 6.17∙10-4 22.0 C 0.21∙10-6 2.00∙10-4 42.90 B Tilt 2.20∙10-3 2.483 1.25∙10-4 5.98∙10-4 23.00 C - 13.00∙10-3 1.52 A Setoxidim 17.0∙10-2 1.832 2.00∙10-6 7.59∙10-4 397.50 C - 11.00∙10-3 8.04 A

**∙10<sup>4</sup> Ty-**

**pe\*\***

*Vmax* **mol/ L s**

**pe mol L–1 mol L–1 \*\***

*S2 Ki*

**∙10<sup>4</sup> Ty-**

3

2

1


1/S1 , 10<sup>3</sup> M-1

0.5

are 1.0‧10–4 and 6‧10–6 mol L–1).

*Vmax* **mol/ L s**

1.0

1/V, 10

6 M-1сек

1/v, 106 M-1·s

1.0‧10–6 mol L–1; *C*NADH = 1.0‧10–4 - 1.5‧10–3 mol L–1.

3.3‧10–4 mol L–1 (*K*<sup>i</sup>

144 Herbicides - Advances in Research

**mol L–1**

**Pesticide** *I***50**

1.5

2.0

2.5

**Figure 7.** Inhibition of the NADH-oxidoreductase with roundup (in the Lineweaver-Burk coordinates) in the absence of the inhibitor (1) and in the presence of 1.17‧10–3 (2) and 2.50‧10–3 mol L–1 (*3*) roundup; *C*NT = 2.467‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NADH = 2.0‧10–4 - 1.5‧10–3 mol L–1.

The dependences of the reciprocal reaction rate on the reciprocal concentration of the NT electron acceptor (with the NADH concentration remaining constant) are shown in Figs 8 and 9. The herbicides lontrel and zenkor (see Fig. 8, Table 3) also inhibit the rate of electron transfer from the NADH-OR active center to NT. The inhibition pattern is uncompetitive (*K*<sup>i</sup> are equal to 7.42‧10–4 and 8.94‧10–4 mol L–1, respectively). The lontrel complex with the copper ion exhibits noncompetitive inhibition, while the complex with cobalt exerts mixed inhibition (see Fig. 9, Table 4).

The Michaelis constants (*K*m) calculated without inhibitors are 6.6‧10–4 and 2.47‧10–3 mol L–1 for NADH and NT, respectively. It was determined in the preliminary experiment that all compounds under study reversibly inhibit NADH-OR.

The data on the effect of other herbicides, fungicides, and lontrel metal complexes on the rate of NADH oxidation and the rate of NT reduction with NADH-oxidoreductase are presented in Table 3.

Of all the compounds studied, the highest inhibitory activities were found for zenkor and basagran (*I*50 are 5.0‧10–4 and 6.0‧10–4 mol L–1, respectively). Lontrel, roundup, tachigaren, and tilt inhibit NADH-OR somewhat less efficient (*I*<sup>50</sup> are 1.1‧10–3, 1.7‧10–3, 2.7‧10–3, and 2.2‧10–3 mol L–1, respectively, see Table 3). Kusagard and setoxidim exhibit weak antireductase activities; they depress the enzyme activity when are present in higher concentrations: 2.7‧10–2 and 1.7‧ 10–2 mol L–1, respectively. In terms of the *K*<sup>i</sup> values with respect to NADH, the herbicides and fungicides can be arranged in the following activity sequence: zenkor > lontrel > basagran > > kusagard > tachigaren > roundup > tilt > setoxidim. This sequence is similar to the sequence of complexation constants of these compounds with NADH given in Table 2.

Lontrel, zenkor, basagran, and roundup inhibit the reduction of NT in a uncompetitive manner, apparently, due to nonspecific interaction with the protein matrix outside the enzyme active center. This interaction could induce conformational changes around the electron transfer site, which result in the inhibition of enzymatic activity. Meanwhile, kusagard, setoxidim, tilt, and tachigaren compete with NT for the binding region on the enzyme. These

**, 10-4 M-1 Type \*** *h*

Cu(CH3COO)2 13.2 0.7 A 1.0 4.0 C (NH4)6Mo7·O24 6.6 4.4 A 1.0 0.4 A Co(CH3COO)2 8.8 0.02 A 1.0 13.1 D Fe(C3H9COO)2 4.4 4.1 A 1.0 11.7 C Ni(CH3COO)2 14.7 0.7 A 1.0 11.7 C Mg(CH3COO)2 no inhibition 3.6 A

Zn(CH3COO)2 no inhibition 2.5 D

Mn(CH3COO)2 no inhibition 22.3 D

**Inhibitor – М(L)<sup>2</sup>**

http://dx.doi.org/10.5772/54679

147

**, 10-4 M-1 Type \***

*Ki*

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

2- does not change the character of the process.

differences can be due to different structures of the pesticides examined.

*S2***, 10-4 М** *Ki*

MgSO4 no inhibition

ZnSO4 no inhibition

MnSO4 no inhibition

replacement of the SO4

In the absence of an inhibitor *Vmax* = 2.8‧10-6 mol L–1 s–1; *S2* = 3.3‧10-4 mol L–1.

\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

2- anion by (СН3СОО)<sup>2</sup>

**Table 4.** Kinetic parameters of inhibition of NADH-oxidoreductase at the artifical electron acceptor NT.

The metal complexes of lontrel are known (Saratovskikh et al., 1988; Saratovskikh et al., 1990) to exhibit herbicide activities *in vivo*. In addition, as noted above, the complex formed by the herbicide lontrel with the copper ion exhibits a much higher inhibitory activity than the starting lontrel. Therefore, we carried out an additional study of a series of complexes of these pesticides with different doubly charged metal ions, M(L)2, and the salts of these metals. As can be seen from the experimental kinetic curves of the dependence of the oxidation rate of NADH-OR on the NADH concentration at a constant NT concentration presented in Fig. 10, the addition of Ni(ac)2 in the concentrations from 3.3‧10-6 to 5‧10-4 М inhibits oxidoreductase functioning and decreases the rate of formation of the reaction product formazan, and at a considerable inhibitor concentration the reaction rate can decrease to zero. Similar studies were carried out with other salts. The dependences of the rate of the enzymatic reaction on the concentration of salt of the metal-inhibitor can be determined from the obtained curves (Fig. 11). It is seen that Mg(II) does not inhibit NADH-OR even at the highest of the concentrations studied, namely, 10-2 М. The Zn(II) and Mn(II) salts also exerted no inhibitory effect, and the

**Inhibitor – solt**

**Figure 8.** Inhibition of the NADH-oxidoreductase with zenkor (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 3.33‧10–4 (2) and 5.00‧10–4 mol L–1 (3) of zenkor; *C*NADH = 0.656‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NT = 7.2‧10–5 - 6.4‧10–3 mol L–1.

**Figure 9.** Inhibition of the NADH-oxidoreductase with zenkor (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 3.33‧10–4 (2) and 5.00‧10–4 mol L–1 (3) of zenkor; *C*NADH = 0.656‧10–3 mol L–1; *C*enzyme = 1.0‧10–6 mol L–1; *C*NT = 7.2‧10–5 - 6.4‧10–3 mol L–1.

Lontrel, zenkor, basagran, and roundup inhibit the reduction of NT in a uncompetitive manner, apparently, due to nonspecific interaction with the protein matrix outside the enzyme active center. This interaction could induce conformational changes around the electron transfer site, which result in the inhibition of enzymatic activity. Meanwhile, kusagard, setoxidim, tilt, and tachigaren compete with NT for the binding region on the enzyme. These differences can be due to different structures of the pesticides examined.


In the absence of an inhibitor *Vmax* = 2.8‧10-6 mol L–1 s–1; *S2* = 3.3‧10-4 mol L–1.

10–2 mol L–1, respectively. In terms of the *K*<sup>i</sup>

146 Herbicides - Advances in Research

1

2

3

1/V, 106 M-1сек

*C*enzyme = 1.0‧10–6 mol L–1; *C*NT = 7.2‧10–5 - 6.4‧10–3 mol L–1.

*C*enzyme = 1.0‧10–6 mol L–1; *C*NT = 7.2‧10–5 - 6.4‧10–3 mol L–1.

1/v, 106 M-1·s

4

5

values with respect to NADH, the herbicides and

3

2

1

fungicides can be arranged in the following activity sequence: zenkor > lontrel > basagran > > kusagard > tachigaren > roundup > tilt > setoxidim. This sequence is similar to the sequence


1/S2, 103 M-1

**Figure 8.** Inhibition of the NADH-oxidoreductase with zenkor (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 3.33‧10–4 (2) and 5.00‧10–4 mol L–1 (3) of zenkor; *C*NADH = 0.656‧10–3 mol L–1;

3

2

1/S2 , 10<sup>3</sup> M-1

1/S2, 103 M-1

1


**Figure 9.** Inhibition of the NADH-oxidoreductase with zenkor (in the Lineweaver-Burk coordinates) in the absence of an inhibitor (1) and in the presence of 3.33‧10–4 (2) and 5.00‧10–4 mol L–1 (3) of zenkor; *C*NADH = 0.656‧10–3 mol L–1;


0.2 0.4 0.6 0.8 1.0 1.2

1/V, 106 M-1сек

1/v, 106 M-1·s

1/S2 , 103 M-1

of complexation constants of these compounds with NADH given in Table 2.

\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

**Table 4.** Kinetic parameters of inhibition of NADH-oxidoreductase at the artifical electron acceptor NT.

The metal complexes of lontrel are known (Saratovskikh et al., 1988; Saratovskikh et al., 1990) to exhibit herbicide activities *in vivo*. In addition, as noted above, the complex formed by the herbicide lontrel with the copper ion exhibits a much higher inhibitory activity than the starting lontrel. Therefore, we carried out an additional study of a series of complexes of these pesticides with different doubly charged metal ions, M(L)2, and the salts of these metals.

As can be seen from the experimental kinetic curves of the dependence of the oxidation rate of NADH-OR on the NADH concentration at a constant NT concentration presented in Fig. 10, the addition of Ni(ac)2 in the concentrations from 3.3‧10-6 to 5‧10-4 М inhibits oxidoreductase functioning and decreases the rate of formation of the reaction product formazan, and at a considerable inhibitor concentration the reaction rate can decrease to zero. Similar studies were carried out with other salts. The dependences of the rate of the enzymatic reaction on the concentration of salt of the metal-inhibitor can be determined from the obtained curves (Fig. 11). It is seen that Mg(II) does not inhibit NADH-OR even at the highest of the concentrations studied, namely, 10-2 М. The Zn(II) and Mn(II) salts also exerted no inhibitory effect, and the replacement of the SO4 2- anion by (СН3СОО)<sup>2</sup> 2- does not change the character of the process.

*V*, 106

Mc-1

v, 106

M·s-1

1/v, 106 M-1·s

**Figure 10.** Kinetic curves of the dependence of the oxidation rate of NADH-oxidoreductase on the NADH concentra‐ tion at a constant concentration of NT. СNT = 6.68‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4 – 1.5‧10-3 М; (1) entry without inhibitors; in the presence of Ni(ас)2 in the concentration (2) 3.3‧10-6 М; (3) 3.0‧10-5 М, and (4) 1.7‧10-4 М.

1/S2, 103 M-1

v, 106

M·s-1

When determining the influence of the metal salts on the reduction of NT, the maximum values


**Figure 12.** Dependences of the reciprocal inhibition reaction rate of NADH-oxidoreductase by Fe(асас)<sup>2</sup> on the inverse NT concentration at a constant concentration of NADH (in the Lineweaver–Burk coordinates); CNT = 1.0‧10-5–7.0‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.43‧10-3 М; (1) without an inhibitor; in the presence of Fe(асас)2 in the concentration

*3*

respectively. The minimum value *Ki* = 2.0‧10-6 М-1 was calculated for Со(II). The inhibition constants of Cu(L)2, Co(L)2, Fe(L)2, and Ni(L)2 are one to three orders of magnitude higher than

lower than that of Мо(II). The phenomena of the appearance of or increase in the inhibitory effect were also observed for the metal complexes with other organic ligands (Tatjanenko et al., 1985).

concentration, 103 M

1/*S2* , 10<sup>4</sup> <sup>M</sup>-1 1/S2, 104 M-1

*2*

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

*1*

The following series can be arranged for an increase in the values of *Ki* with the change in the NT concentration for the metal salts: Со(II) < Cu(II) ~ Ni(II) < Fe(II) ~ Mo(VI). A similar regularity is observed for the lontrel complexes in the activity sequence by the values of *Ki*

Mo(L)2 < Zn(L)2 < Mg(L)2 < Cu(L)2 < Ni(L)2 = Fe(L)2 < Co(L)2 < Mn(L)2; the electron-donor properties of the metal exert a strong effect on the strength of the bond between the inhibitor

The results of the influence of (ML2) on the enzymatic activity of NADH-OR are presented in Table 4. On going from the salt to complexes, the type of inhibition changes completely: from the same competitive type for all metal salts to several different variants, namely, mixed for Co(L)2 and uncompetitive for Ni(L)2, Fe(L)2, and Cu(L)2. Only Mo(VI) and Мо(L)2 retain one, competitive, type of inhibition. The Mg(II), Zn(II), and Mn(II) ions do not inhibit NADH-OR, whereas Mg(L)2 compete with an electron acceptor for the binding site on the enzyme and Zn(L)2 and Mn(L)2 have the mixed type of inhibition with respect to both NT and NADH. Interestingly, as shown above, Mg(L)2, Zn(L)2, and Mn(L)2, in turn, do not interact with NADH. The formation constants of the M(L)2–NADH complexes (*К*c/f: Fe < Mo < (Co) < Cu < Ni) show a direct correlation with the inhibition constants of NADH-OR by an electron donor for both

= 4.4‧10-4 М-1 and 4.1‧10-4 М-1,

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149

1/S2, 103 M-1

an order of magnitude

:

for the inhibition constants were found for Mo(VI) and Fe(II): *Ki*

1/v, 106 M-1·s


(2) 3.0‧10-4 М and (3) 5.0‧10-4 М.

1/*V*, 10<sup>6</sup>

 M-1 с

and enzyme.

those for the corresponding salts. On the contrary, Mo(L)2 showed *Ki*

the complexes and metal salts *KiM+*: Co < Ni < Cu < Mo < Fe (Table 5).

1/S2, 103 M-1 **Figure 11.** Dependences of the enzymatic reaction rate on the concentration of the metal salts: (1) Mg(ас)2; (2) Мо(аm)6; (3) Fe(асас)2. CNT = 6.68‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4–1.5‧10-3 М.

The dependences of the reciprocal rate of the reductase reaction on the inverse concentration of the artificial electron accetor (NT) at a fixed NADH concentration for Fe(асас)2 are presented in Fig. 12. The considered metal salts inhibit the reduction of NT, competing with the artificial electron acceptor for the binding region with NADH-OR. As follows from Table 4, the corresponding complexes, except for Мо(L)2, do not compete with the electron acceptor.

16

1/*V*, 10<sup>6</sup>

 M-1 с

**Figure 12.** Dependences of the reciprocal inhibition reaction rate of NADH-oxidoreductase by Fe(асас)<sup>2</sup> on the inverse NT concentration at a constant concentration of NADH (in the Lineweaver–Burk coordinates); CNT = 1.0‧10-5–7.0‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.43‧10-3 М; (1) without an inhibitor; in the presence of Fe(асас)2 in the concentration (2) 3.0‧10-4 М and (3) 5.0‧10-4 М.

When determining the influence of the metal salts on the reduction of NT, the maximum values for the inhibition constants were found for Mo(VI) and Fe(II): *Ki* = 4.4‧10-4 М-1 and 4.1‧10-4 М-1, respectively. The minimum value *Ki* = 2.0‧10-6 М-1 was calculated for Со(II). The inhibition constants of Cu(L)2, Co(L)2, Fe(L)2, and Ni(L)2 are one to three orders of magnitude higher than those for the corresponding salts. On the contrary, Mo(L)2 showed *Ki* an order of magnitude lower than that of Мо(II). The phenomena of the appearance of or increase in the inhibitory effect were also observed for the metal complexes with other organic ligands (Tatjanenko et al., 1985). 1/S2, 103 M-1 concentration, 103 M

The following series can be arranged for an increase in the values of *Ki* with the change in the NT concentration for the metal salts: Со(II) < Cu(II) ~ Ni(II) < Fe(II) ~ Mo(VI). A similar regularity is observed for the lontrel complexes in the activity sequence by the values of *Ki* : Mo(L)2 < Zn(L)2 < Mg(L)2 < Cu(L)2 < Ni(L)2 = Fe(L)2 < Co(L)2 < Mn(L)2; the electron-donor properties of the metal exert a strong effect on the strength of the bond between the inhibitor and enzyme.

1/v, 106 M-1·s

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 *V*, 106

0.0

0.2

0.4

0.6

v, 106

M·s-1

148 Herbicides - Advances in Research

0.8

1.0

1.2

1.4

*V*, 106

Mc-1

Mс-1

0.0 0.5 1.0 1.5 2.0 2.5

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

**Figure 10.** Kinetic curves of the dependence of the oxidation rate of NADH-oxidoreductase on the NADH concentra‐ tion at a constant concentration of NT. СNT = 6.68‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4 – 1.5‧10-3 М; (1) entry without inhibitors; in the presence of Ni(ас)2 in the concentration (2) 3.3‧10-6 М; (3) 3.0‧10-5 М, and (4) 1.7‧10-4 М.

**Figure 11.** Dependences of the enzymatic reaction rate on the concentration of the metal salts: (1) Mg(ас)2; (2)

The dependences of the reciprocal rate of the reductase reaction on the inverse concentration of the artificial electron accetor (NT) at a fixed NADH concentration for Fe(асас)2 are presented in Fig. 12. The considered metal salts inhibit the reduction of NT, competing with the artificial electron acceptor for the binding region with NADH-OR. As follows from Table 4, the corresponding complexes, except for Мо(L)2, do not compete with the electron acceptor.

Мо(аm)6; (3) Fe(асас)2. CNT = 6.68‧10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4–1.5‧10-3 М.

*3 2*

Концентрация солей металлов, 10<sup>3</sup>

*1*

*S1* , 103 M

*4*

*3*

*2*

*1*

concentration, 10 М3 M

v, 106

M·s-1

1/v, 106 M-1·s

1/S2, 103 M-1

1/S2, 103 M-1

v, 106

M·s-1

The results of the influence of (ML2) on the enzymatic activity of NADH-OR are presented in Table 4. On going from the salt to complexes, the type of inhibition changes completely: from the same competitive type for all metal salts to several different variants, namely, mixed for Co(L)2 and uncompetitive for Ni(L)2, Fe(L)2, and Cu(L)2. Only Mo(VI) and Мо(L)2 retain one, competitive, type of inhibition. The Mg(II), Zn(II), and Mn(II) ions do not inhibit NADH-OR, whereas Mg(L)2 compete with an electron acceptor for the binding site on the enzyme and Zn(L)2 and Mn(L)2 have the mixed type of inhibition with respect to both NT and NADH. Interestingly, as shown above, Mg(L)2, Zn(L)2, and Mn(L)2, in turn, do not interact with NADH. The formation constants of the M(L)2–NADH complexes (*К*c/f: Fe < Mo < (Co) < Cu < Ni) show a direct correlation with the inhibition constants of NADH-OR by an electron donor for both the complexes and metal salts *KiM+*: Co < Ni < Cu < Mo < Fe (Table 5).


М, Mо(L)<sup>2</sup> *I50* = 8.5‧10-4 М; *i.e.*, the antireductase activity of the metal ion is threefold higher than that of the corresponding metal complex with lontrel. The order of increasing *I50* (*i.e.*, decreasing the antireductase activity) in the case of the complexes Cu(L)2 < Mo(L)2 < Zn(L)2 < Fe(L)<sup>2</sup> = L < Co(L)2 < Ni(L)2 = Mg(L)2 < Mn(L)<sup>2</sup> is reciprocal to that presented above for the salts

The highest of the calculated inhibition constants with respect to the electron donor (NADH) belongs to Fe(II) and Mo(VI), and the values of *Ki* are14.2‧10-4 М-1 and 8.8‧10-4 М-1, respectively.

The character of inhibition of NADH-OR with copper acetate at different substrate (NADH) concentrations at a fixed concentration of NT is shown in Fig. 13. The intersection of the straight lines in one point but not in the axis indicates that the inhibition follows the so-called mixed type. The same character of inhibition was demonstrated by Mo(аm)6 and Ni(ас)2. The Cu(L)2, Mo(L)2, and Fe(L)<sup>2</sup> complexes compete with NADH for the binding site on NADH-OR (Table 5). The strength of the bond between Cu(L)2 and NADH-OR is nearly 20 times higher than that


**Figure 13.** Dependences of the reciprocal inhibition reaction rate of NADH-oxidoreductase by Cu(ас)2 on the inverse NT concentration at a constant concentration of NT (in the Lineweaver–Burk coordinates). CNT = 6.68 10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4–1.5‧10-3 М; (1) without an inhibitor; in the presence of Cu(ас)2 in the concentration (2) 3.0‧

orders of magnitude on going from the salt to the cobalt complex: 0.014‧10-4 and 3.8‧10-4 М-1, respectively. However, Со(ас)2 inhibits oxidoreductase manifesting the noncompetitive

On the contrary, the strength of the bond between Ni(L)<sup>2</sup> and NADH-OR decreases: *Ki*

= 6.0‧10-6 М-1 and 1.2‧10-4 М-1, respectively. A similar situation was observed for

*3*

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

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151

*2*

*1*

1/S2, 103 M-1

= 0.9‧

1/*S1* , 103 M-1

1/S1, 103 M-1

concentration, 103 M

= 12.4‧10-4 М-1 for Ni(L)2. The inhibition constant increases by two

= 1.4‧10-6 М-1) was determined for Co(II).

of the same metals.

The lowest value (*Ki*

of Cu(II); *Ki*

10-5 М and (3) 1.7‧10-4М.

10-4 М-1 for Ni(II) and *Ki*

v, 106

M·s-1

molybdenum and iron and their complexes.

1

2

3

4

1/v, 106 M-1·s

5

6

1/*V*, 106

 M-1 с

In the absence of an inhibitor *Vmax* = 2.8‧10-6 mol L–1 s–1; *S2* = 3.3‧10-4 mol L–1.

\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

**Table 5.** Kinetic parameters of inhibition of NADH-oxidoreductase at the substrate NADH.

Among the lontrel complexes with metal ions, only the complexes with Mg and Mo proved to be competitive reductase inhibitors with respect to NT. The lontrel complexes with Cu, Ni, and Fe ions inhibit the enzyme noncompetitively, while the lontrel complexes with Mn, Zn, and Co display a mixed type of inhibition.

Table 5 demonstrates the kinetic parameters of inhibition of NADH-oxidoreductase with respect to the substrate NADH. The highest inhibition ability is shown by Co(II): *I*50 = 1.33‧ 10-6 М. The copper and nickel salts exhibits the equal values: *I*50 = 3.3‧10-5 М. The weak antireductase activity was demonstrated by Fe(II) and Mo(VI), namely, *I50* = 3.3‧10-4 М. The metal salts can be arranged in the following series by the calculated values of *I50*: Co(ас)2 < Ni(ас)2 = Cu(ас)2 < Fe(асас)2 = Mo(аm)6.

The inhibitory effect of the complexes ZnL2, CoL2, FeL2, and MoL2 is similar to that of the parent lontrel (*I*50 are 1.0‧10–3, 1.5‧10–3, 1.1‧10–3, and 0.85‧10–3 mol L–1, respectively), while the inhibitory effects of MgL2, MnL2, and NiL2 are much lower.

The Mg, Zn, Co, and Ni complexes with lontrel exhibit noncompetitive inhibition with respect to NADH, whereas Mn, Cu, Fe, and Mo complexes inhibit the enzyme competitively. The difference between the inhibition patterns may be related to the difference between the acceptor abilities of the metal ions.

A comparison of the data presented in Tables 4 and 5 shows that the metal salts inhibit the enzyme in much lower concentrations. The value of *I50* of a salt is one to three orders of magnitude lower than that of the complexes of the corresponding metal. The maximum difference in the values of *I50* is observed for Со(II) and Co(L)2: 1.33‧10-6 and 1.5‧10-3 М, respectively. The minimum difference is characteristic of molybdenum: Mo(VI) *I50* = 3.3‧10-4 М, Mо(L)<sup>2</sup> *I50* = 8.5‧10-4 М; *i.e.*, the antireductase activity of the metal ion is threefold higher than that of the corresponding metal complex with lontrel. The order of increasing *I50* (*i.e.*, decreasing the antireductase activity) in the case of the complexes Cu(L)2 < Mo(L)2 < Zn(L)2 < Fe(L)<sup>2</sup> = L < Co(L)2 < Ni(L)2 = Mg(L)2 < Mn(L)<sup>2</sup> is reciprocal to that presented above for the salts of the same metals.

**Inhibitor – solt**

150 Herbicides - Advances in Research

*I50***, М**

MgSO4 no inhibition

ZnSO4 no inhibition

MnSO4 no inhibition

and Co display a mixed type of inhibition.

Ni(ас)2 = Cu(ас)2 < Fe(асас)2 = Mo(аm)6.

acceptor abilities of the metal ions.

effects of MgL2, MnL2, and NiL2 are much lower.

In the absence of an inhibitor *Vmax* = 2.8‧10-6 mol L–1 s–1; *S2* = 3.3‧10-4 mol L–1.

\* The type of inhibition: A - competitive, B - uncompetitive, C - noncompetitive, D - mixed.

**Table 5.** Kinetic parameters of inhibition of NADH-oxidoreductase at the substrate NADH.

*Vmax***, Мс-1**

*S1***, 10-4 М**

*Ki* **, 104 M-1 Type\***

Cu(CH3COO)2 3.3·10-5 1.1·10-6 3.9 1.2 D 3.3·10-4 0.06 A (NH4)6Mo7·O24 3.3·10-4 1.4·10-6 3.0 8.8 D 8.5·10-4 0.1 A Со(CH3COO)2 1.3·10-6 9.7·10-7 1.2 0.01 B 1.5·10-3 13.7 C Fe(C3H9COO)2 3.3·10-4 4.6 14.2 A 1.1·10-3 1.1 A Ni(CH3COO)2 3.3·10-5 7.7·10-7 3.2 0.9 D 2.0·10-3 12.4 C Mg(CH3COO)2 no inhibition 2.0·10-3 12.7 C

Zn(CH3COO)2 no inhibition 1.0·10-3 10.2 C

Mn(CH3COO)2 no inhibition 3.0·10-3 3.8 A

Among the lontrel complexes with metal ions, only the complexes with Mg and Mo proved to be competitive reductase inhibitors with respect to NT. The lontrel complexes with Cu, Ni, and Fe ions inhibit the enzyme noncompetitively, while the lontrel complexes with Mn, Zn,

Table 5 demonstrates the kinetic parameters of inhibition of NADH-oxidoreductase with respect to the substrate NADH. The highest inhibition ability is shown by Co(II): *I*50 = 1.33‧ 10-6 М. The copper and nickel salts exhibits the equal values: *I*50 = 3.3‧10-5 М. The weak antireductase activity was demonstrated by Fe(II) and Mo(VI), namely, *I50* = 3.3‧10-4 М. The metal salts can be arranged in the following series by the calculated values of *I50*: Co(ас)2 <

The inhibitory effect of the complexes ZnL2, CoL2, FeL2, and MoL2 is similar to that of the parent lontrel (*I*50 are 1.0‧10–3, 1.5‧10–3, 1.1‧10–3, and 0.85‧10–3 mol L–1, respectively), while the inhibitory

The Mg, Zn, Co, and Ni complexes with lontrel exhibit noncompetitive inhibition with respect to NADH, whereas Mn, Cu, Fe, and Mo complexes inhibit the enzyme competitively. The difference between the inhibition patterns may be related to the difference between the

A comparison of the data presented in Tables 4 and 5 shows that the metal salts inhibit the enzyme in much lower concentrations. The value of *I50* of a salt is one to three orders of magnitude lower than that of the complexes of the corresponding metal. The maximum difference in the values of *I50* is observed for Со(II) and Co(L)2: 1.33‧10-6 and 1.5‧10-3 М, respectively. The minimum difference is characteristic of molybdenum: Mo(VI) *I50* = 3.3‧10-4

**Inhibitor – М(L)<sup>2</sup>**

*, 10-4* **M-1 Type\***

v, 106

M·s-1

*I50***, М** *Ki*

The highest of the calculated inhibition constants with respect to the electron donor (NADH) belongs to Fe(II) and Mo(VI), and the values of *Ki* are14.2‧10-4 М-1 and 8.8‧10-4 М-1, respectively. The lowest value (*Ki* = 1.4‧10-6 М-1) was determined for Co(II).

The character of inhibition of NADH-OR with copper acetate at different substrate (NADH) concentrations at a fixed concentration of NT is shown in Fig. 13. The intersection of the straight lines in one point but not in the axis indicates that the inhibition follows the so-called mixed type. The same character of inhibition was demonstrated by Mo(аm)6 and Ni(ас)2. The Cu(L)2, Mo(L)2, and Fe(L)<sup>2</sup> complexes compete with NADH for the binding site on NADH-OR (Table 5). The strength of the bond between Cu(L)2 and NADH-OR is nearly 20 times higher than that of Cu(II); *Ki* = 6.0‧10-6 М-1 and 1.2‧10-4 М-1, respectively. A similar situation was observed for molybdenum and iron and their complexes.

1/*V*, 106

 M-1 с

**Figure 13.** Dependences of the reciprocal inhibition reaction rate of NADH-oxidoreductase by Cu(ас)2 on the inverse NT concentration at a constant concentration of NT (in the Lineweaver–Burk coordinates). CNT = 6.68 10-3 М; Cenzyme = 2.83‧10-7 М; CNADH = 1.0‧10-4–1.5‧10-3 М; (1) without an inhibitor; in the presence of Cu(ас)2 in the concentration (2) 3.0‧ 10-5 М and (3) 1.7‧10-4М.

On the contrary, the strength of the bond between Ni(L)<sup>2</sup> and NADH-OR decreases: *Ki* = 0.9‧ 10-4 М-1 for Ni(II) and *Ki* = 12.4‧10-4 М-1 for Ni(L)2. The inhibition constant increases by two orders of magnitude on going from the salt to the cobalt complex: 0.014‧10-4 and 3.8‧10-4 М-1, respectively. However, Со(ас)2 inhibits oxidoreductase manifesting the noncompetitive

concentration, 103 M

1/S2, 103 M-1

pounds. Apparently, the combination of these factors is responsible for the different mecha‐

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

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153

The Hill factor (*h*) is nearly equal or close to 2 for all of the compounds, which indicates that two inhibitor molecules can be attached simultaneously to the enzyme both inside and probably outside the active center (Table 3). This parameter substantially distinguishes pesticides and metal complexes from metal salts. The Hill factor for the considered metal salts is close to unit (Table 5), indicating the possibility of the addition of only one metal cation to

Evidently, the metal cation tends to negatively charged groups of amino acids, namely, carboxyl, sulfide, and amide groups. In addition, Mn, Mg, and Zn for the least stable complexes with carbonyl, and Ni and Fe form the strongest complexes with this group (Holtzclaw & Collman, 1957; Isatt et al., 1954), which agrees with our data. Thus, the interaction of this kind near the active center of the enzyme can result in conformational changes in the region of electron transfer, which is manifested as noncompetitive and mixed types of inhibition. It is known (Tsuprun et al., 1987; Fitzpatrick et al., 2005) that the 2Fe-2S cluster is in the composition of the active center of NADH-OR. The change in the character of inhibition on going from divalent metal salts to their lontrel complexes can additionally be due to the interaction of the complexes not only with the protein matrix but also with the metal of the 2Fe-2S cluster.

Figure 15 represents the scheme of direction of the inhibitor attack. The intramolecular elec‐ tron transfer in the active center of NADH-OR proceeds from flavine adenine dinucleotide (FAD) to the iron-sulfur cluster 2Fe-2S and further to an artificial electron acceptor (Bayer et al., 1996; Du et al., 2000b; Ganson & Jensen, 1988). For competitive inhibition, the cation occupies the site of NT and this breaks the chain of electron transfer. The fact that the metal salts have much lower *I50* compared to the complexes indicates easiness of this interaction. The determining factor in

N

Cl

**+**

NADH

FAD

2e-

Cl

C O

OH

the behavior of the metal salts is the structure of the electronic shells of the cation.

M(L)2

e-

M2+

INT+

Fe

S-

S

**Figure 15.** Scheme of direction of the attack from different inhibitors.

Fe

nisms of NADH-OR inhibition by the considered compounds.

the enzyme.

**Figure 14.** EPR of the metal complex (1) CuL2 = 5.0‧10–4 mol L–1; (2) CuL2 =5.0‧10–4 mol L–1 in the presence of NADH = 6.0‧10–4 mol L–1; (3) CuL2 = 5.0‧10–4 mol L–1 in the presence of NADH = 6.0‧10–4 mol L–1 and enzyme. L is lontrel. The conditions of measurements: 77 К, microwave 10 mW, magnetic field modulation 0.4 mT.

character of inhibition for NADH. At the same time, the Со(L)2 complex does not compete with NADH for the binding site on the enzyme.

Of the compounds considered, only Fe(асас)2 competes with an electron acceptor for the binding site in the active center of the enzyme. The same character of inhibition is retained in the iron complex with respect to NADH, but *Ki* for Fe(L)<sup>2</sup> is 14 times lower than that for Fe(II): 1.1‧10-4 and 14.2‧10-4 М-1, respectively.

The change in the pattern of inhibition by the lontrel complexes with doubly charged metal ions may be due to the fact that the interaction of these complexes with the protein involves other protein ligands (thiol groups, the imidazole part of histidine, and other amino acid residues of the peptide chain). In addition, the metal ions in these complexes can be reduced by the enzyme, as it was shown by ESR for the lontrel complexes with copper ions (Fig. 14).

Our results indicate that the herbicides, fungicides, and lontrel metal complexes can react with NADH-OR in the cavity of the protein matrix in which either NADH binding or electron transfer to a natural or artificial electron acceptor takes place. An additional interaction of these compounds beyond the enzyme active center cannot also be dismissed. The structure, size, and spatial configuration of the pesticide molecule are also significant. Apparently, the large size of kusagard and sedoxidim molecules prevents them from entering the cavity of the protein globule, which may account for the weak inhibition of NADH-OR by these com‐ pounds. Apparently, the combination of these factors is responsible for the different mecha‐ nisms of NADH-OR inhibition by the considered compounds.

The Hill factor (*h*) is nearly equal or close to 2 for all of the compounds, which indicates that two inhibitor molecules can be attached simultaneously to the enzyme both inside and probably outside the active center (Table 3). This parameter substantially distinguishes pesticides and metal complexes from metal salts. The Hill factor for the considered metal salts is close to unit (Table 5), indicating the possibility of the addition of only one metal cation to the enzyme.

Evidently, the metal cation tends to negatively charged groups of amino acids, namely, carboxyl, sulfide, and amide groups. In addition, Mn, Mg, and Zn for the least stable complexes with carbonyl, and Ni and Fe form the strongest complexes with this group (Holtzclaw & Collman, 1957; Isatt et al., 1954), which agrees with our data. Thus, the interaction of this kind near the active center of the enzyme can result in conformational changes in the region of electron transfer, which is manifested as noncompetitive and mixed types of inhibition. It is known (Tsuprun et al., 1987; Fitzpatrick et al., 2005) that the 2Fe-2S cluster is in the composition of the active center of NADH-OR. The change in the character of inhibition on going from divalent metal salts to their lontrel complexes can additionally be due to the interaction of the complexes not only with the protein matrix but also with the metal of the 2Fe-2S cluster.

Figure 15 represents the scheme of direction of the inhibitor attack. The intramolecular elec‐ tron transfer in the active center of NADH-OR proceeds from flavine adenine dinucleotide (FAD) to the iron-sulfur cluster 2Fe-2S and further to an artificial electron acceptor (Bayer et al., 1996; Du et al., 2000b; Ganson & Jensen, 1988). For competitive inhibition, the cation occupies the site of NT and this breaks the chain of electron transfer. The fact that the metal salts have much lower *I50* compared to the complexes indicates easiness of this interaction. The determining factor in the behavior of the metal salts is the structure of the electronic shells of the cation.

**Figure 15.** Scheme of direction of the attack from different inhibitors.

character of inhibition for NADH. At the same time, the Со(L)2 complex does not compete with

**Figure 14.** EPR of the metal complex (1) CuL2 = 5.0‧10–4 mol L–1; (2) CuL2 =5.0‧10–4 mol L–1 in the presence of NADH = 6.0‧10–4 mol L–1; (3) CuL2 = 5.0‧10–4 mol L–1 in the presence of NADH = 6.0‧10–4 mol L–1 and enzyme. L is lontrel. The

conditions of measurements: 77 К, microwave 10 mW, magnetic field modulation 0.4 mT.

Of the compounds considered, only Fe(асас)2 competes with an electron acceptor for the binding site in the active center of the enzyme. The same character of inhibition is retained in

The change in the pattern of inhibition by the lontrel complexes with doubly charged metal ions may be due to the fact that the interaction of these complexes with the protein involves other protein ligands (thiol groups, the imidazole part of histidine, and other amino acid residues of the peptide chain). In addition, the metal ions in these complexes can be reduced by the enzyme, as it was shown by ESR for the lontrel complexes with copper ions (Fig. 14). Our results indicate that the herbicides, fungicides, and lontrel metal complexes can react with NADH-OR in the cavity of the protein matrix in which either NADH binding or electron transfer to a natural or artificial electron acceptor takes place. An additional interaction of these compounds beyond the enzyme active center cannot also be dismissed. The structure, size, and spatial configuration of the pesticide molecule are also significant. Apparently, the large size of kusagard and sedoxidim molecules prevents them from entering the cavity of the protein globule, which may account for the weak inhibition of NADH-OR by these com‐

for Fe(L)<sup>2</sup> is 14 times lower than that for Fe(II):

NADH for the binding site on the enzyme.

152 Herbicides - Advances in Research

the iron complex with respect to NADH, but *Ki*

1.1‧10-4 and 14.2‧10-4 М-1, respectively.

Present in the composition of the complex, the metal cannot act as a free cation, since it is significantly affected by the ligand environment. The considered ligand (lontrel) is capable of occupying the site of NADH, donor of two electrons, in the active center of the enzyme. Probably, the interaction with iron of the cluster occurs through the carboxyl of the ligand and due to a high electron density of chloropyridine. As a result, the ligand or complex inhibits the NADH-binding region of the electron-transfer chain.

10-3 10-2 10-1

**Figure 16.** Change in the toxicity of the pesticides towards "luminescent" bacteria *Beneckea harveyi vs.* concentration of the pesticide in the concentration range from 10-1 to 10-3 М. (1) zenkor; (2) lontrel; (3) roundup; (4) basagran; (5)

The results of measurements of toxicity of herbicides and fungicide tachigaren with respect to luminescent bacteria *Beneckea harveyi* are presented in Fig. 16. The plots show that the toxicity of solutions increases proportionally to the concentration with an increase in the pesticide concentration. However, the rates of toxicity increase differ for different substances: the rate of zenkor is considerably higher than those of other compounds, whereas for tachigaren the toxicity coefficient (Т, %) increases with the lowest rate. Zenkor has the highest toxicity of all the compounds evaluated. Lontrel, roundup, and basagran differ from each other to a lower extent. The lowest toxicity was determined to be tachigaren. In fact, even at the highest of the studied concentrations, 10-1 М, tachigaren remains to be a nontoxic compound with Т < 19%. In the concentration range from 3‧10-3 to 10-1 М basagran exhibits a weak toxicity: 24 < Т ≤ 40%. In the concentration range from 10-3 to 3‧10-3 М, all compounds (except zenkor) are lowly toxic with Т ≤ 50%. Beginning from the concentrations 10-3 М (zenkor), 10-2 М (lontrel), and 10-1 М

The results of measurements of pesticide toxicity are presented in Table 6. Parameter ЕС<sup>50</sup> corresponds to the toxicant concentration resulting in the 50% decrease in the luminescence of bacteria. The values of EC50 increase in the order zenkor < lontrel < roundup < basagran < tachigaren. An analysis of the data in Table 6 shows that the pesticides are arranged in the decrease in toxicity in the same sequence, which is retained at all concentrations studied.

It was discussed previously that the metal complexes of herbicide lontrel are characterized by a considerable antireductase activity. However, there are no literature data on the quantitative estimation of their toxicity towards hydrobionts. The results of the study of the influence on *Beneckea harveyi* are given in Fig. 17. It is seen that all the metal complexes are toxic even at a

*1*

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

http://dx.doi.org/10.5772/54679

155

*2*

*3*

*5*

**C**, M

C, M

1/S2, 103 M-1

concentration, 103 M

(roundup), they become highly toxic compounds.

*4*

20

tachigaren.

40

60

80

100

120

140

*Т***,%**

T, %

We have previously reported (Aliev et al., 1988) that the lontrel complexes can exist in solution in both the dimeric and polymeric forms. The structures of the complexes allow them to play the role of both the electron-donor and electron-acceptor. It can be assumed that the complexes form polymer chains in which the ligands acts as a "bridge" and the metal of the complex pulls electrons of the 2Fe-2S cluster of the active center of the enzyme. The existence of two binding sites in the active center likely explains the fact that *Ki* of the complex (in the case of Мо, with respect to the electron acceptor; in the case of Сu, Mo, and Fe, with respect to the electron donor) is one to two orders of magnitude lower than that of the salt. 1/v, 106 M-1·s

Obviously, the evaluated compounds, herbicides, fungicides, and metal complexes of herbi‐ cide lontrel, can retard oxidation processes in plants and living organisms. However, the effect of these compounds on various components of the ecosystem is manifested in a diverse character of reactions of the organisms, in the multiphase character of these reactions, and in the possible transition of one effect to another. To analyze the influence of the studied compounds on the living organisms, we chose hydrobionts – marine "luminescent" bacteria *Benechea harveyi* (strain В 1 7 – 667F), (Zhmur & Orlova, 2007; Kuz`mich et al., 2002). It is validly considered (Tsvetkov & Konichev, 2006) that hydrobionts are the most appropriate objects for the study of biochemical adaptations to the toxic action and, as a consequence, they are the most popular laboratory test-objects and object-indicators for calibrations of contaminations under natural conditions. On the other hand, when the effect of chemical toxicants on biological systems is studied, the time of the toxicological analysis itself compared to the rate of formation of metabolites in the living organism is significant. Bacteria *Beneckea harveyi* allow researchers to obtain a fast response and to compare their effect on the change in the enzymatic (luciferase) activity. It is important that the inactivation of only one enzyme is controlled *in vitro* on a model system.

Present in the composition of the complex, the metal cannot act as a free cation, since it is significantly affected by the ligand environment. The considered ligand (lontrel) is capable of occupying the site of NADH, donor of two electrons, in the active center of the enzyme. Probably, the interaction with iron of the cluster occurs through the carboxyl of the ligand and due to a high electron density of chloropyridine. As a result, the ligand or complex inhibits the

NADH-binding region of the electron-transfer chain.

154 Herbicides - Advances in Research

N

donor) is one to two orders of magnitude lower than that of the salt.

system.

Cl

Cl

C

O

S O

We have previously reported (Aliev et al., 1988) that the lontrel complexes can exist in solution in both the dimeric and polymeric forms. The structures of the complexes allow them to play the role of both the electron-donor and electron-acceptor. It can be assumed that the complexes form polymer chains in which the ligands acts as a "bridge" and the metal of the complex pulls electrons of the 2Fe-2S cluster of the active center of the enzyme. The existence of two binding sites in the active center likely explains the fact that *Ki* of the complex (in the case of Мо, with respect to the electron acceptor; in the case of Сu, Mo, and Fe, with respect to the electron

Obviously, the evaluated compounds, herbicides, fungicides, and metal complexes of herbi‐ cide lontrel, can retard oxidation processes in plants and living organisms. However, the effect of these compounds on various components of the ecosystem is manifested in a diverse character of reactions of the organisms, in the multiphase character of these reactions, and in the possible transition of one effect to another. To analyze the influence of the studied compounds on the living organisms, we chose hydrobionts – marine "luminescent" bacteria *Benechea harveyi* (strain В 1 7 – 667F), (Zhmur & Orlova, 2007; Kuz`mich et al., 2002). It is validly considered (Tsvetkov & Konichev, 2006) that hydrobionts are the most appropriate objects for the study of biochemical adaptations to the toxic action and, as a consequence, they are the most popular laboratory test-objects and object-indicators for calibrations of contaminations under natural conditions. On the other hand, when the effect of chemical toxicants on biological systems is studied, the time of the toxicological analysis itself compared to the rate of formation of metabolites in the living organism is significant. Bacteria *Beneckea harveyi* allow researchers to obtain a fast response and to compare their effect on the change in the enzymatic (luciferase) activity. It is important that the inactivation of only one enzyme is controlled *in vitro* on a model

OR

N Cl Cl

Cu O Fe OR

1/v, 106 M-1·s

C O

O

**Figure 16.** Change in the toxicity of the pesticides towards "luminescent" bacteria *Beneckea harveyi vs.* concentration of the pesticide in the concentration range from 10-1 to 10-3 М. (1) zenkor; (2) lontrel; (3) roundup; (4) basagran; (5) tachigaren.

1/S2, 103 M-1 concentration, 103 M The results of measurements of toxicity of herbicides and fungicide tachigaren with respect to luminescent bacteria *Beneckea harveyi* are presented in Fig. 16. The plots show that the toxicity of solutions increases proportionally to the concentration with an increase in the pesticide concentration. However, the rates of toxicity increase differ for different substances: the rate of zenkor is considerably higher than those of other compounds, whereas for tachigaren the toxicity coefficient (Т, %) increases with the lowest rate. Zenkor has the highest toxicity of all the compounds evaluated. Lontrel, roundup, and basagran differ from each other to a lower extent. The lowest toxicity was determined to be tachigaren. In fact, even at the highest of the studied concentrations, 10-1 М, tachigaren remains to be a nontoxic compound with Т < 19%. In the concentration range from 3‧10-3 to 10-1 М basagran exhibits a weak toxicity: 24 < Т ≤ 40%. In the concentration range from 10-3 to 3‧10-3 М, all compounds (except zenkor) are lowly toxic with Т ≤ 50%. Beginning from the concentrations 10-3 М (zenkor), 10-2 М (lontrel), and 10-1 М (roundup), they become highly toxic compounds.

The results of measurements of pesticide toxicity are presented in Table 6. Parameter ЕС<sup>50</sup> corresponds to the toxicant concentration resulting in the 50% decrease in the luminescence of bacteria. The values of EC50 increase in the order zenkor < lontrel < roundup < basagran < tachigaren. An analysis of the data in Table 6 shows that the pesticides are arranged in the decrease in toxicity in the same sequence, which is retained at all concentrations studied.

It was discussed previously that the metal complexes of herbicide lontrel are characterized by a considerable antireductase activity. However, there are no literature data on the quantitative estimation of their toxicity towards hydrobionts. The results of the study of the influence on *Beneckea harveyi* are given in Fig. 17. It is seen that all the metal complexes are toxic even at a concentration of 10-7 М. The toxicity increases linearly with an increase in the concentration for all complexes. Curves 1–4 corresponding to the complexes of different metals are parallel, indicating the same rate of toxicity increasing on the concentration of ML2. At all concentrations the toxicity coefficients of the complexes decrease in the series CuL2 > CoL2 > MnL2 > MgL2. As follows from Table 6, EC50 measured by the "probit analysis" method (Loshadkin et al., 2002) change in the same order: CuL2 > CoL2 > NiL2 > MoL2 > MnL2 > ZnL2 > L > MgL2. It should especially be mentioned that the toxicity of almost all lontrel complexes with respect to *Beneckea harveyi* turned out higher than that of the starting herbicide. The value of ЕС50 for CuL2 is more than two orders of magnitude lower and that for ZnL2 is four orders of magnitude lower than that for lontrel. The exclusion is the MgL2 complex, whose toxicity is insignificantly lower than that of the starting lontrel.

A comparison of the data in Table 6 shows that metal complexe toxicity is manifested at concentrations two orders of magnitude lower than that of any pesticide considered. Similarly, the EC50 parameters of the pesticides and metal complexes indicate thatthe values of toxicity of the latter are two orders of magnitude higher than that of all pesticides and, particularly, herbicide lontrel. In the range of the concentrations studied, both the pesticides and the

Kinetics and Mechanism of Inhibition of Oxidation Enzymes by Herbicides

The study performed of the toxicity of herbicides, fungicides, and metal complexes of lontrel towards bacteria *Beneckea harveyi* showed high toxicity. This fact indicates the antiluciferase activity of these compounds. The determined values of EC50 correlate with both the complex‐

Thus, despite the substantial differences in the chemical structures, all of the herbicides, fungicides and lontrel metal complexes studied inhibit NADH-OR at both the electron-donor and the electron-acceptor sites. These compounds inhibit the NADH-binding region and, perhaps, the intramolecular electron transfer from FAD to the 2Fe-2S iron-sulfur cluster and further to an artificial electron acceptor. This conclusion is consistent with the published data on the interruption of the electron transfer chain (Tissut et al., 1984; Macherel et al., 1982; Higgins et al., 1981) by pesticides and the involvement of metals in this process (Bayer et al., 1996; Du et al., 2000b; Ganson & Jensen 1988). The character of inhibition changes on going from the metal salts to their complexes: all the metal salts compete with an electron acceptor for the binding site, and the complexes compete with an electron donor. It is very important that in some cases, of Мо, Сu, and Fe, the strength of the bond with the enzyme increases on going from the metal ion to the ligand and to the complex (М+2 < L < ML2), which should result in an increase in the toxic properties of the complexes compared to the metals and pesticides.

The enzyme NADH-OR is abundant in nature and is found in both unicellular and multicel‐ lular organisms; therefore, broad-scale practical use of herbicides and fungicides may entail

Evidently, one of the mechanisms of formation of toxicity of herbicides seems to be inhibition of redox processes in organisms of different trophic levels. In birds and mammals (including people), the inhibition of the oxidative enzymes decreases the protective functions of the organism and results in various maladies. Moreover, almost all well understood diseases of modern man are caused by a poor ecological state of the environment (Gichev, 2003; Klyush‐

Several thousands of pesticides are produced in the world. About 180 pesticides are widely used (Mel'nikov, 1987). Maximum allowable concentrations are substantiated only for 30 of them (Fomin, 2000). The assertion about an exclusive importance of their use for the enhance‐ ment of agricultural productivity is not substantiated (Yablokov, 1990; Fisher et al., 2002;

their accumulation in living organisms and severe environmental consequences.

nikov, 2005; Mogush, 1984; Isaev, 1997; Lisichkin & Chernov, 2003).

) of enzyme

157

http://dx.doi.org/10.5772/54679

complexes of seven metals are arranged in the same sequence by toxicity decreasing.

ation constants (K) of these compounds with NADH and inhibition constants (*K*<sup>i</sup>

NADH-OR by these compounds.

**4. Conclusion**


**Table 6.** Toxicity values (ЕC50) for the pesticides and lontrel metal complexes (ML2) measured by the *Beneckea harveyi* biotest.

**Figure 17.** Change in the toxicity index determined on "luminescent" bacteria *Beneckea harveyi vs.* logarithm of the concentration of the metal complexes: (1) CuL2; (2) CoL2; (3) MnL2; (4) MgL2.

A comparison of the data in Table 6 shows that metal complexe toxicity is manifested at concentrations two orders of magnitude lower than that of any pesticide considered. Similarly, the EC50 parameters of the pesticides and metal complexes indicate thatthe values of toxicity of the latter are two orders of magnitude higher than that of all pesticides and, particularly, herbicide lontrel. In the range of the concentrations studied, both the pesticides and the complexes of seven metals are arranged in the same sequence by toxicity decreasing.

The study performed of the toxicity of herbicides, fungicides, and metal complexes of lontrel towards bacteria *Beneckea harveyi* showed high toxicity. This fact indicates the antiluciferase activity of these compounds. The determined values of EC50 correlate with both the complex‐ ation constants (K) of these compounds with NADH and inhibition constants (*K*<sup>i</sup> ) of enzyme NADH-OR by these compounds.

### **4. Conclusion**

concentration of 10-7 М. The toxicity increases linearly with an increase in the concentration for all complexes. Curves 1–4 corresponding to the complexes of different metals are parallel, indicating the same rate of toxicity increasing on the concentration of ML2. At all concentrations the toxicity coefficients of the complexes decrease in the series CuL2 > CoL2 > MnL2 > MgL2. As follows from Table 6, EC50 measured by the "probit analysis" method (Loshadkin et al., 2002) change in the same order: CuL2 > CoL2 > NiL2 > MoL2 > MnL2 > ZnL2 > L > MgL2. It should especially be mentioned that the toxicity of almost all lontrel complexes with respect to *Beneckea harveyi* turned out higher than that of the starting herbicide. The value of ЕС50 for CuL2 is more than two orders of magnitude lower and that for ZnL2 is four orders of magnitude lower than that for lontrel. The exclusion is the MgL2 complex, whose toxicity is insignificantly lower than

**ML2**

1. Zenkor (4.4±0.1)·10-3 0.94 CuL2 (1.3±0.1)·10-5 0.0058 2. Lontrel (8.0±0.3)·10-3 1.54 CoL2 (3.0±0.2)·10-4 0.13 3. Roundup (2.0±0.1)·10-2 3.38 NiL2 (5.0±0.2)·10-4 0.22 4. Basagran (2.9±0.1)·10–2 7.01 MoL2 (7.0±0.3)·10-4 0.33 5. Tachigaren (1.0±0.2)·10-1 9.91 MnL2 (1.6±0.1)·10-3 0.69 6. ZnL2 (2.0±0.1)·10-3 0.89 7. MgL2 (1.0±0.1)·10-2 4.06

**Table 6.** Toxicity values (ЕC50) for the pesticides and lontrel metal complexes (ML2) measured by the *Beneckea harveyi*

**Figure 17.** Change in the toxicity index determined on "luminescent" bacteria *Beneckea harveyi vs.* logarithm of the

concentration of the metal complexes: (1) CuL2; (2) CoL2; (3) MnL2; (4) MgL2.

**М g/l М g/l**

**EC50**

**EC50**

that of the starting lontrel.

156 Herbicides - Advances in Research

**№ Pesticide**

biotest.

Thus, despite the substantial differences in the chemical structures, all of the herbicides, fungicides and lontrel metal complexes studied inhibit NADH-OR at both the electron-donor and the electron-acceptor sites. These compounds inhibit the NADH-binding region and, perhaps, the intramolecular electron transfer from FAD to the 2Fe-2S iron-sulfur cluster and further to an artificial electron acceptor. This conclusion is consistent with the published data on the interruption of the electron transfer chain (Tissut et al., 1984; Macherel et al., 1982; Higgins et al., 1981) by pesticides and the involvement of metals in this process (Bayer et al., 1996; Du et al., 2000b; Ganson & Jensen 1988). The character of inhibition changes on going from the metal salts to their complexes: all the metal salts compete with an electron acceptor for the binding site, and the complexes compete with an electron donor. It is very important that in some cases, of Мо, Сu, and Fe, the strength of the bond with the enzyme increases on going from the metal ion to the ligand and to the complex (М+2 < L < ML2), which should result in an increase in the toxic properties of the complexes compared to the metals and pesticides.

The enzyme NADH-OR is abundant in nature and is found in both unicellular and multicel‐ lular organisms; therefore, broad-scale practical use of herbicides and fungicides may entail their accumulation in living organisms and severe environmental consequences.

Evidently, one of the mechanisms of formation of toxicity of herbicides seems to be inhibition of redox processes in organisms of different trophic levels. In birds and mammals (including people), the inhibition of the oxidative enzymes decreases the protective functions of the organism and results in various maladies. Moreover, almost all well understood diseases of modern man are caused by a poor ecological state of the environment (Gichev, 2003; Klyush‐ nikov, 2005; Mogush, 1984; Isaev, 1997; Lisichkin & Chernov, 2003).

Several thousands of pesticides are produced in the world. About 180 pesticides are widely used (Mel'nikov, 1987). Maximum allowable concentrations are substantiated only for 30 of them (Fomin, 2000). The assertion about an exclusive importance of their use for the enhance‐ ment of agricultural productivity is not substantiated (Yablokov, 1990; Fisher et al., 2002; Yudanova, 1989; Khan, 1980; Moses, 1988; Paasivitra, 1988). The use of pesticides is an example for gaining a short-term profit of chemical companies owing to the long-term detriment for all society (Skurlatov et al., 1994; Yablokov, 1990; Suley & Uilcoks, 1983; Kurdyukov, 1982).

[10] Dixon, M. & Webb, E.C. (1979). *Enzymes*. Longman Group Ltd., L.–N. Y.–Toronto.

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[11] Dorval, J.; Leblond, V.S. & Hontela, A. (2003). Oxidative stress and loss of cortisol se‐ cretion in adrenocortical cells of rainbow trout (*Oncorhynchus mykiss*) exposed in vi‐ tro to endosulfan, an organochlorine pesticide. *Aquat. toxicol*. Vol. 63. No. 3, pp.

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### **Author details**

#### E. A. Saratovskikh

Institute of Problem of Chemical Physics, Russian Academia of Science, Russia

### **References**


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**Author details**

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E. A. Saratovskikh

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**Chapter 8**

**Involvement of Lignin-Modifying Enzymes in the**

The high demand for food due to the increase in the world population has led to an increasing use of plant protection products, also known as pesticides, in order to improve productivity. However, along with the success in food production, the accumulation of these persistent chemicals in soil and water is harmful to the environmental and human health [1]. In recent years, agricultural pesticide application has increased all over the world. A total of 5,197 mil lbs of pesticides were used worldwide in 2007 [2]. There are many different types of pesticides; each is meant to be effective against specific pests. The term "-cide" comes from the Latin word "to kill." Among them, herbicides account for the largest market (around 40%) share, followed by insecticides (17%) and fungicides (10%). Herbicides are chemicals used to kill undesired plants, such as weeds, and they are used extensively in home gardens and in agriculture. Due to the relative predominance of herbicides, the present review will focus on this class of

Herbicides have well defined pros and cons associated with their use. Their use tends to increase yields, and thus makes a significant difference in food production, particularly in countries that struggle periodically against famines. On the other hand, they can cause water pollution when erosion and/or rainwater carry the chemicals off the farms together with the eroded soils after each rainfall. Herbicides vary in their potential to persist in the soil. They are chemically heterogeneous and their structure is one of the main features that determine persistence. For example, some substitutions on aromatic rings (-F, -Cl, -NO2, -NH2, -CF3, -

> © 2013 Coelho-Moreira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Coelho-Moreira et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Degradation of Herbicides**

Jaqueline da Silva Coelho-Moreira, Giselle Maria Maciel, Rafael Castoldi,

http://dx.doi.org/10.5772/55848

**1. Introduction**

compounds.

Simone da Silva Mariano, Fabíola Dorneles Inácio,

Adelar Bracht and Rosane Marina Peralta

Additional information is available at the end of the chapter
