The Interaction of Ferric Ions with Jack Bean Urease by Isothermal Titration Calorimetry

Abstract

The aim of the investigations was to measure the influence of Fe³⁺ ion on jack bean urease (JBU) activity. Interaction between Fe³⁺ and JBU, is examined using isothermal titration calorimetry. It was found that Fe³⁺ ions acted as a non-cooperative inhibitor of JBU, and there is a set of 12 identical and independent binding sites for Fe³⁺ ions. The small structural parameters show that there are little changes on the JBU structure, indicating that Fe³⁺ has minor effect on the JBU activity. The association equilibrium constant is 42,484.13 ± 110 mol l⁻¹, indicating the moderate interaction of Fe³⁺ ion with JBU. The molar enthalpy of binding is ΔH = −4.33 kJ mol⁻¹.

Introduction

There are a variety of nitrogenous fertilizers available in the market; however, urea consumption is 38 %, which is higher than other nitrogenous fertilizers due to the relatively low manufacturing cost and high concentration of N [1]. Jack bean urease (JBU) rapidly catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. The product, ammonia, of such decomposition reactions diffuses across the cytoplasmic membrane, buffering the periplasmic space and allows growth in the presence of extracellular gastric acid and responsible for negative effects of urease activity in human health, such as causing peptic ulcers and stomach cancer. Besides, in agriculture the efficiency of soil nitrogen fertilization with urea decreases due to ammonia volatilization and root damage caused by soil pH increase [2–4].

Therefore, it is interesting to control the activity of urease through the use of its inhibitors in order to counteract these negative effects in medicine, environmental and agronomic. Heavy metal ions inhibit both plant and bacterial urease at the following approximate order of effectiveness: Hg²⁺ ≈ Ag⁺ > Cu²⁺ > Ni²⁺ > Cd²⁺ > Zn²⁺ > Co²⁺ > Fe³⁺ > Pb²⁺ > Mn²⁺ with Hg²⁺, Ag⁺ and Cu²⁺ ions practically known as the strongest inhibitors [3–6]. The objective of this study was to assess the urease activity and conformational changes of JBU due to its binding to Fe³⁺ ion.

Materials and Methods

Jack bean urease (JBU; MW = 545.34 kDa), Tris salt and Fe³⁺ ions obtained from sigma chemical Co. The isothermal titration microcalorimetric experiments were performed with the four channel commercial microcalorimetric system. Fe³⁺ 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 Fe³⁺ solution into the perfusion vessel was repeated 28 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 Fe³⁺ 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

The obtain results were reported in Table 1 and shown graphically in Fig. 1.

Table 1 The heats of Fe³⁺+JBU interaction at 300 K in 30 mmol l⁻¹ Tris buffer solution of pH = 7

Fig. 1

Comparison between the experimental heats (filled triangle) at 300 K, for Fe³⁺ + JBU interactions and the calculated data (lines) via Eq. 1. The [Fe³⁺]/μM are the concentrations of [Fe(NO₃)₃] solution in μmol l⁻¹

We have shown previously that the heats of the ligand + JBU interactions in the aqueous solvent mixtures can be calculated via the following equation [7–12]:

$$ q = q_{\hbox{max} } x^{\prime}_{B} - \delta_{A}^{\theta } (x^{\prime}_{A} L_{A} + x^{\prime}_{B} L_{B} ) - (\delta_{B}^{\theta } - \delta_{A}^{\theta } )(x^{\prime}_{A} L_{A} + x^{\prime}_{B} L_{B} )x^{\prime}_{B} $$

(1)

q is the heat of Fe³⁺ + JBU interaction and the optimized value of q _max represents the heat value upon occupation of all binding sites on JBU. The parameters $\delta_{A}^{\theta }$ and $\delta_{B}^{\theta }$ are the indexes of JBU stability in the low and high Fe³⁺ concentrations, respectively. 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. $x^{\prime}_{B}$ can be expressed as follows:

$$ x^{\prime}_{B} = \frac{{px_{B} }}{{x_{A} + px_{B} }} $$

(2)

One can express x _B fractions, as the Fe³⁺ concentrations divided by the maximum concentration of the Fe³⁺ upon saturation of all JBU as follows:

$$ x_{B} = \frac{{[Fe^{3 + } ]}}{{[Fe^{3 + } ]_{\hbox{max} } }},\quad x_{A} = 1 - x_{\begin{subarray}{l} B \\ \end{subarray} } $$

(3)

[Fe³⁺] is the concentration of Fe³⁺ and [Fe ³⁺]_max is the maximum concentration of the Fe³⁺ upon saturation of all 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 Fe³⁺ in the buffer solution, q _dilut, as follows:

$$ L_{A} = q_{\text{dilut}} + x_{B} \left( {\frac{{\partial q_{\text{dilut}} }}{{\partial x_{B} }}} \right), L_{B} = q_{\text{dilut}} + x_{A} \left( {\frac{{\partial q_{\text{dilut}} }}{{\partial x_{B} }}} \right) $$

(4)

The heats of Fe³⁺+JBU interactions, q, were fitted to Eq. 1 across the whole Fe³⁺ compositions. In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Fig. 1). $\delta_{A}^{\theta }$ and $\delta_{B}^{\theta }$ 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 extended solvation model. The binding parameters for Fe³⁺+JBU interactions recovered from Eq. 1 were listed in Table 2. 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.

Table 2 Binding parameters for JBU + Fe³⁺ interactions

For a non-cooperative interaction:

$$ \frac{{q_{{{\text{max}}}} - q}}{{q_{{{\text{max}}}} }}[{\text{JBU}}] = \left( {\frac{{q_{{{\text{max}}}} - q}}{q}} \right)[Fe^{{3 + }} ]\frac{1}{g} - \frac{{K_{d} }}{g}. $$

(5)

[JBU] and [Fe ³⁺] are concentrations of JBU and Fe ³⁺, respectively. q represents the heat value at a certain Fe ³⁺ ion concentration and the optimized values for q _max represents the heat value upon saturation of all JBU. Checking different values for q _max, the best linear plot of $(\frac{{q_{\hbox{max} } - q}}{{q_{\hbox{max} } }})[{\text{JBU}}]$ versus $(\frac{{q_{\hbox{max} } - q}}{q})[Fe^{3 + } ]$was approached as follows:

$$ \frac{{ - 3465 - q}}{{ - 3465}}[JBU] = \left(\frac{{ - 3465 - q}}{q}\right)[Fe^{{3 + }} ]\frac{1}{{12}} - \frac{{1.95}}{{12}}. $$

(6)

Comparing Eqs.  5 and 6, the number of binding sites on JBU (g = 12) and the dissociation equilibrium constant (K _d = 1.95 μmol l⁻¹) can be calculated. Dividing the optimized q _max amount of −3465 μJ (equal to −52.32 kJ mol⁻¹) by g = 12, gives ΔH = −4.34 ± 0.09 kJ mol⁻¹.

The change in standard Gibbs free energy (ΔG°) can be calculated according to the equation (7), which its value can use in equation (8) for calculating the change in standard entropy (ΔS°) of binding process.

$$ \Updelta G = - RT\ln K_{a} $$

(7)

$$ \Updelta G = \Updelta H - T\Updelta S $$

(8)

where K _a is the association binding constant (K _a  = 1/K _d ). The obtained value for K _a is 42,484.13 ± 110 L mol⁻¹ Hence:

ΔG = −26.58 ± 0.09 kJ mol⁻¹ΔS = 0.07 ± 0.01 kJ mol⁻¹ K⁻¹

All thermodynamic parameters for the interaction between JBU and Fe³⁺ ion have been summarized in Table 2. The small and negative value of $\delta_{A}^{\theta }$ indicates that Fe³⁺ is a poor inhibitor of JBU activity.