Catalytic studies of glutathione transferase from Clarias gariepinus (Burchell) in dilute and crowded solutions

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Highlights

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    Polyethylene glycol 6000 enhanced the catalytic activity of GST from Clarias gariepinus.

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    Ficoll 70 and BSA caused a decrease in catalytic activity of GST from Clarias gariepinus.

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    A mixture of the three crowders decreased the catalytic activity of GST from Clarias gariepinus.

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    Mechanism of catalysis in dilute and crowded solutions was steady-state random sequential.

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    Crowding agents altered the catalytic activity of GST from Clarias gariepinus but has no effect on its mechanism of action.

Abstract

Kinetic properties of purified Clarias gariepinus glutathione transferase (CgGST) was studied in the presence of Ficoll 70, Polyethylene glycol (PEG) 6000, bovine serum albumin (BSA) and in dilute solution. This was done to mimic the cytosol thereby unraveling the actual mechanism of detoxication involving glutathione transferase (GST) in the crowded intracellular milieu. CgGST from the liver of Clarias gariepinus was purified to homogeneity by affinity chromatography on glutathione (GSH) - agarose. Initial-velocity study was performed by varying the concentrations of GSH at various fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) and vice-versa. Data obtained were fitted to the three equations representing random-ordered, compulsory-ordered and ping-pong mechanisms to obtain kinetic parameters. Product inhibition studies using sodium chloride (NaCl) was done by varying the concentrations of NaCl and CDNB at a fixed concentration of GSH and vice-versa. Data obtained were fitted to three equations representing competitive, non-competitive and uncompetitive inhibitions to obtain the inhibition constants (KiGSH and KiCDNB). Optimal temperature of CgGST activity was 20 °C both in dilute and crowded solutions. Maximum velocity (Vmax) in dilute solution was decreased, while KmGSH and KmCDNB were increased in the presence of the crowding agents. Turnover number (kcat), catalytic efficiency - kcat/KmGSH, kcat/KmCDNB and inhibition constants – (KiGSH and KiCDNB) were reduced in crowded solutions. Mechanism of catalysis was steady – state random sequential in both dilute and crowded solutions. The study concluded that although the catalytic efficiency of the enzyme was reduced in crowded solution, mechanism of catalysis remains the same in both crowded and dilute solutions.

Introduction

The intracellular environment which is the natural compartment of most enzymes that catalyze myriad of biochemical reactions is usually enriched with diverse macromolecules whose total concentration could exceed 300 g/l (Cayley et al., 1991; Zimmerman and Trach, 1991; Conlon and Raff, 2003; Zeskind et al., 2007; Cheung et al., 2013). Despite this fact, most enzymes that have been so far purified and characterized kinetically (with a few exceptions), were assayed in dilute solutions which are completely different from the crowded cytosol. Therefore, findings from such studies may not be a true representation of enzyme kinetics in the natural intracellular milieu. Consequently, efforts geared towards understanding the effect of other non-enzymatic components of the cytosol on the steady state kinetics of selected enzymes have commenced over four decades ago (Laurent, 1971). Predicting the effects of macromolecular crowding agents on steady state kinetics of enzymes in vivo is often a herculean task. Hence, chemically inert synthetic polymers are often introduced into enzyme assays as crowding agents. Frequently used crowding agents include ficoll (Wenner and Bloomfield, 1999; Homchaudhuri et al., 2006; Jiang and Guo, 2007; Pozdnyakova and Wittung-Stafshede, 2010) dextran, (Pozdnyakova and Wittung-Stafshede, 2010; Pastor et al., 2014; Balcells et al., 2014; Poggi and Slade, 2015; Schneider et al., 2015; Wilcox et al., 2016; Fodeke, 2019), polyvinylpyrrolidone (PVP) (Schneider et al., 2015; Poggi and Slade, 2015) and polyethylene glycol (PEG) (Zimmerman and Harrison, 1987; Sasaki et al., 2006; Totani et al., 2008; Aumiller et al., 2014). Reports have suggested that crowding agents can either increase, decrease or show no effect on steady-state kinetics of enzymes (Crowley et al., 2008; Wang et al., 2011; Sarkar et al., 2013a, Sarkar et al., 2013b; Monteith et al., 2015; Cohen and Pielak, 2016, Cohen and Pielak, 2017; Gorensek-Benitez et al., 2017). Several biological reactions and processes have been reported to be influenced by macromolecular crowding. Specifically, studies have shown that crowding significantly affects the catalytic efficiency of DNA ligase (Zimmerman and Pheiffer, 1983), enterobactin-specific isochorismate synthase (Laurent, 1971) and Ras (member of the class of small GTPases) (Minton, 1981). Moreover, thermal stability of cytochrome c (Minton and Wif, 1981), apoflavo-doxin (Pozdnyakova and Wittung-Stafshede, 2010) and creatine kinase (Schneider et al., 2015) was significantly enhanced in the presence of some synthetic crowders. Besides the aforementioned, effects of crowding agents on catalytic proteins have been investigated in a number of housekeeping enzymes including glucose-6-phosphate dehydrogenase, glucose isomerase and carbonic anhydrase (Monterroso and Minton, 2007; Norris and Malys, 2011). However, there is little or no information on investigations regarding detoxication enzymes generally, and glutathione transferase in particular. It was reasoned that glutathione transferase (GST), a major phase II detoxication enzyme from the liver of catfish (Clarias gariepinus) would be a good model to study this, partly because catfish are hardy and thrive in polluted ponds, rivers or streams better than other species of fish. This ability has been attributed in part to the presence of high concentrations of GST in the organism (Ojopagogo et al., 2013). Till date, all studies on CgGST were conducted in dilute buffer solutions which do not represent the cytosolic environment of the enzyme. Therefore, there is a need to study the physicochemical and kinetic properties of the enzyme in crowded solutions that would mimic the intracellular environment so as to provide information on the effect of crowding on the mechanism of detoxication involving GST in Clarias gariepinus.

Section snippets

Materials

Reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), glutathione-agarose, 2-mercaptoethanol, Polyethylene glycol 6000, Ficoll 70, bovine serum albumin (BSA), sodium phosphate dibasic (Na2HPO4), anhydrous sodium phosphate monobasic (NaH2PO4), N,N,N′,N′-tetramethylethylenediamine (TEMED) were obtained from Sigma Chemical Company, St Louis, Mo, USA. DEAE-Tris-acryl was a product of LKB, France. All other reagents were of analytical grade.

Preparation of liver homogenate of C. gariepinus

Adult African catfish (Clarias gariepinus) of

Kinetic parameters of CgGST in dilute and crowded solutions

The kinetic parameters obtained from initial velocity studies (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5(a and b) and product inhibition studies (Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10(a and b) of GST from the liver of C. gariepinus were as shown in Table 1. The double reciprocal plots of the data obtained from the initial velocity studies both in dilute and crowded solutions displayed set of lines that converged to the left of the 1/Vo axis (Figs. 1(a)-5(b)).

When NaCl was used as product

Discussion

Catalytic properties of glutathione transferase from the liver of C. gariepinus was studied in dilute and crowded environments. These properties, usually determined by established kinetic parameters of such enzymes, can provide useful information about their rates and mechanism of catalysis. In the present study, the maximum velocity (Vmax) of CgGST in dilute solution (Table 1) was higher than what was previously reported by Ojopagogo et al. (2013) from cultured juvenile catfish. This may be

Conclusion

The study concluded that catalytic efficiency of CgGST is reduced in the presence of mixed crowding agents probably because of reduction in affinity of the enzyme for substrates, the mechanism of catalysis however remained the same in both dilute and crowded solutions.

Declaration of competing interest

There is no conflict of interest in any form.

Acknowledgement

Authors acknowledge the financial support of TetFund (2015–2016 intervention year), Ekiti State University Ado Ekiti, Ekiti State, Nigeria.

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