Introduction

Jack Bean Urease is found in plants, fungi and bacteria and has the historical interest of being the first enzyme to be crystallized [1]. Urea is a major nitrogenous waste product of biological actions. In general, urea is short-lived and rapidly metabolized by microbial activities [2].

Urease catalyzes the hydrolysis of urea yielding ammonium carbamate. The ammonium carbamate product is unstable and spontaneously degrades to CO₂ and two molecules of ammonia [3].

CO (NH₂)₂ + H₂O→ NH₂COONH₄ → 2NH₃ + CO₂

This reaction leads to high–volatilization losses of ammonia if urea is surface applied. Such ammonia losses will occur particularly on soils poor in sorption capacity, without plant cover and with a high pH [4, 5]. It can also cause severe germination and seedling damage due to ammonia and nitrite (NO₂^–) when the amount placed near the seed is too large [6]. The protonization of ammonia to ammonium leads to a slight rise in pH value (NH₃+ H₂O ➞ NH₄⁺ + OH^–) [7].

Possibly the best way to reduce ammonia production is to slow or stop the conversion of urea to ammonium. Compounds that inhibit the enzymatic breakdown of nitrogenous compounds present in feces and urine can decrease ammonia production. Based on the values of the overall inhibition constant K_i^*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg²⁺> Cu²⁺> Zn²⁺> Cd²⁺> Ni²⁺ > Pb²⁺ > Co²⁺ > Fe³⁺ > As³⁺. Urease inhibitors can block thehydrolysis of urinary urea to ammonium and thus decrease ammonia production [8]. Ammonia lost to the atmosphere may be deposited on land or water causing eutrophication and acidification. Urease inhibitors by delaying ammonia formation and subsequent nitrification can reduce the nitrate content in plants and improve the nutritional quality of vegetables and fodder plants. Urease inhibitors have been very important in farming applications where slowing the conversion of urea to ammonium provides further time for the crops to take up the ammonium. The objective of this study was to assess the urease activity and conformational changes of JBU due to its binding to Cr³⁺ ion.

Materials and method

Jack bean urease (JBU; MW=545.34 kDa), Tris salt and Cr³⁺ ions obtained from sigma chemical Co. The isothermal titration microcalorimetric experiments were performed with the four channel commercial microcalorimetric system. Cr³⁺ solution (4 mmol.L^-1) was injected by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL JBU (37 µmol.L^-1). Injection of Cr³⁺ solution into the perfusion vessel was repeated 27 times, with 10 µL per injection. The calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system. The heat of each injection was calculated by the ‘‘Thermometric Digitam 3’’ software program. The heat of dilution of the Cr³⁺ solution was measured as described above except JBU was excluded. The microcalorimeter was frequently calibrated electrically during the course of the study.

Results and discussion

We have shown previously that the heats of the ligand + JBU interactions in the aqueous solvent systems, can be calculated via the following equation [9-14]:

          

 

q is the heat of Cr³⁺ + JBU interaction and q_max represents the heat value upon saturation of all JBU. The parameters  and  are the indexes of JBU stability in the low and high Cr³⁺ concentrations respectively. Cooperative binding requires that the macromolecule has more than one binding site, since cooperativity results from the interactions between identical binding sites with the same ligand. If the binding of a ligand at one site increases the affinity for that ligand at another site, then the macromolecule exhibits positive cooperativity. Conversely, if the binding of a ligand at one site lowers the affinity for that ligand at another site, then the enzyme exhibits negative cooperativity. If the ligand binds at each site independently, the binding is non-cooperative. p >1 or p <1 indicate positive or negative cooperativity of a macromolecule for binding with a ligand, respectively; p = 1 indicates that the binding is non-cooperative.  can be expressed as follows:

                                                                             

 

We can express  fractions, as the total Cr³⁺ concentrations divided by the maximum concentration of the Cr³⁺ upon saturation of all JBU as follows:

 

[Cr³⁺] is the concentration of Cr³⁺ and [Cr³⁺]_max is the maximum concentration of the Cr³⁺ upon saturation of all JBU. In general, there will be “g” sites for binding of Cr³⁺ per JBU molecule and ν is defined as the average moles of bound Cr³⁺ per mole of total JBU. L_A and L_B are the relative contributions due to the fractions of unbound and bound metal ions in the heats of dilution in the absence of JBU and can be calculated from the heats of dilution of Cr³⁺ in the buffer solution, q_dilut, as follows:

 

The heats of Cr³⁺+JBU interactions, q, were fitted to Eq. 1 across the whole Cr³⁺ compositions. In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Fig. 1). The optimized  and  values are recovered from the coefficients of the second and third terms of Eq. 1. The small relative standard coefficient errors and the high r² values (0.99999) support the method. The binding parameters for Cr³⁺+JBU interactions recovered from Eq. 1 were listed in Table 1. The agreement between the calculated and experimental results (Fig. 1) is striking, and gives considerable support to the use of Eq. 1.  and  values for Cr³⁺+JBU interactions is negative, indicating that in the low and high concentrations of the metal ions the JBU structure is destabilized, resulting in an decrease in its activity. Destabilization by a ligand indicates that the ligand binds preferentially (either at more sites or with higher affinity) to the unfolded (denatured) enzyme or to a partially folded intermediate form of the enzyme. Such effects are characteristic of nonspecific interactions, in that the nonspecific ligand binds weakly to many different groups at the protein, so that binding becomes a function of ligand concentration, which is increased through unfolding events. p=1 indicates that the binding is non-cooperative.

According to the recently data analysis method, a plot of  versus should be a linear plot by a slope of 1/g and the vertical-intercept of  , which g and K_d can be obtained [15-20].

 

 

 

Where g is the number of binding sites, K_d is the dissociation equilibrium constant, M₀ and L₀ are concentrations of JBU and Cr³⁺, respectively, , q represents the heat value at a certain Cr³⁺ ion concentration and q_max represents the heat value upon saturation of all JBU. If q and q_max are calculated per mole of JBU then the molar enthalpy of binding for each binding site (DH) will be DH= q_max/g. Dividing the q_max amount of 12100 µJ (equal to 181.68 kJmol^-1)  by g=12, therefore, gives DH= 15.10 kJmol^-1.

To compare all thermodynamic parameters in metal binding process for JBU, the change in standard Gibbs free energy (DG^◦) should be calculated according to the equation (6), which its value can use in equation (7) for calculating the change in standard entropy (DS^◦) of binding process.

                                                                                                              (6)

 

Where K_a is the association binding constant (the inverse of the dissociation binding constant, K_d). The K_a value are obtained 6.79×10⁶ L.mol^-1 Hence:

DG^◦ = −39.23 kJ mol^-1  DS^◦ = 0.18 kJ mol^-1 K^-1

All thermodynamic parameters for the interaction between JBU and Cr³⁺ ion have been summarized in Table 1. All thermodynamic parameters of the complex formation including ΔG°, ΔH°, ΔS°, indicate that the process is endothermic and entropy driven. This issue shows the predominant role of hydrophobic forces in interaction between Cr³⁺ and JBU. The large association equilibrium constant of the Cr³⁺+JBU complex, indicates that chromium is strongly associated with JBU. The results show, that Cr³⁺ ions caused inhibition of urease activity significantly. even more than Hg²⁺ as We have reported previously that  and  values for Hg²⁺+JBU interaction are -0.52 and 1.81 [8]. Thereby, we can conclude that Cr³⁺ ions can inhibit JBU more than Hg²⁺ ions.

Figure 1: Comparison between the experimental heats (○) at 300 K, for Cr3+ + JBU interactions and the calculated data (lines) via Eq. 1. The [Cr3+] are the concentrations of [Cr (NO3)3] solution in μmol.L−1   Click here to View figure

Table 1: Binding parameters for JBU+ Cr³⁺ interactions.  p=1 indicates that the binding is non-cooperative. The negative  and  values prove that the JBU+Cr³⁺ complexes are not stable, indicating that Cr³⁺ inhibit the JBU activity significantly. The large association equilibrium constant indicates a strong interaction of chromium with JBU.

parameters	T=300K
	6.79×106±220
	1±0.01
	-4.43±0.13
	-9.01±0.15
	15.10±0.09
	-39.23±0.07
	0.18±0.02

Conclusion

The binding parameters for JBU+ Cr³⁺ interactions, indicate that the binding is non-cooperative. The negative   and  values prove that the JBU+Cr³⁺ complexes are not stable, indicating that Cr³⁺ inhibit the JBU activity significantly. The large association equilibrium constant indicates a strong interaction of  Cr³⁺ ion with JBU.

Acknowledgements

The financial support Islamic azad university of takestan is gratefully acknowledged.

References

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