A Molecular Modeling Study of the Urease Active Site

*e-mail: bruno@antas.agraria.uniss.it

Abstract

Urease catalyzes the decomposition of urea to ammonium and carbamate ions by its active site that contains two nickel(II) atoms. Here we report the results of a Molecular Dynamics investigation performed on a system constituted by the cavity of the enzyme and one hydroxamic acid molecule which acts as an inhibitor of the protein. The results well agree with experimental data and represent a valid starting point to design more efficient urease inhibitors.

Key words: Urease inhibitors, Molecular Dynamics, hydroxamates

Introduction

Urease is an enzyme that is present in many plants and in soil. It catalyzes the hydrolysis of urea to ammonium and carbamate ions, which decompose to carbon dioxide and ammonia. The active site contains two nickel(II) atoms which, as shown by X-ray analysis, are linked by a carbamate bridge; furthermore, two imidazole nitrogen atoms are bound to each nickel atom, and a carboxylate group and a water molecule fill the remaining coordination site of the metal ion¹. Among the known inhibitors of urease, the most efficient are phosphorodiamides, phosphorotriamides² and hydroxamic acid derivatives³.

In order to discriminate among the inhibition capacities of various compounds, it is important to understand the coordination mechanism between the active site of the enzyme and the inhibitor. The molecular modeling techniques consitute a valid and powerful tool to investigate such systems, as proved by the numerous papers dealing with such subjects^4-6. Alternatives docking modes of the urea on urease active site have been studied² which gave information about the reaction mechanism of the inhibitors.

In order to achieve a better knowledge of the urease inhibition mechanism we performed a Molecular Dynamics (MD) study on the formation of the hydroxamate complexes with urease. The results, compared with a recent model⁷ proposed to explain the inhibition of urease, show a satisfactory agreement.

Materials and Methods

The urease active site and the hydroxamic acid molecule were used as a model inhibitor are shown in Fig.1. The conformation of the inhibitor was determined by a Molecular Mechanics search for the minimum energy conformation, while the coordinates of the urease active site were taken from the Brookhaven Protein Data Bank (PDB), ID=1kau¹. As no information about the proton positions were given in the PDB structure, in order to keep equal to zero the site total charge, we held that two imidazole rings of the binding site were in a protonate form, while the remaining two in a -1 charged deprotonated form. Each nickel atom coordinates one protonated and one deprotonated imidazole ring. One water molecule completes the nichel coordination spere. The coordination geometry of the first nickel atom is pseudo tetrahedral, while that of the second is roughly trigonal bipyramidal.

The MD simulations were performed using the DLPOLY2⁸ program. The AMBER⁹ and UFF¹⁰ force fields were used with the necessary adaptations. The partial atomic charges were calculated by fitting the electrostatic potential computed by ab initio HF-SCF calculations at the 6-311G** accuracy level with MP2 perturbation treatment^11,12. The Gamess program¹³ was employed to perform both the ab initio and charge fitting computations. The values of the fitted charges are shown in Fig.2.

The MD simulations were performed at T = 298 K, with no periodic condition applied, as we were concerned only in studying the docking of the acid to the enzyme. The atoms of the active site were held rigid, while those of the inhibitor molecule were free to move, as well as those of the water molecule. The cut-off for Coulomb forces was set to 20 Å, so that all interactions were included. Several trajectories were generated allowing the hydroxamic acid molecule to start from different positions, with the carbonyl oxygen, the oxime oxygen and the methyl group oriented towards the axis joining the nickel atoms, 20 Å far away. Each trajectory was equilibrated for 50 ps with a time step of 0.001 ps, then a 2 ns run was performed for accumulated data, even if the final configurations were reached after 100 ps, and took about 30000 sec on a IBM RS 6000 Mod. 355.

Results and discussion

A snapshot of the urease - hydroxamic acid couple after 2 ns trajectory is shown in Fig.3.

The hydroxamic acid acts as a monodentate ligand, being bound to the nickel atom in a pseudo tetrahedral coordination geometry by the carbonyl oxygen. This conformation agrees completely with the models proposed by Stemmler⁶ and Lippard¹⁴ for urea bound to nickel (see Scheme). In both cases the hydroxamic acid binds the nickel atom in the same way.

We performed several MD experiments changing the initial position and orientation of the hydroxamic acid. In all the cases we obtained the final conformation shown in Fig.3.

The second step in the Scheme involves a deprotonated hydroxamate bridge between the nickel atoms which acts as a chelate on the first nickel atom. In order to reproduce these alkaline conditions, we removed an hydrogen atom from the hydroxamic acid and calculated the partial atom charges again (see Fig.2). The initial conformation for this new set of MD simulations is shown in Fig.3. After a 2 ns run we observed the final conformation represented in Fig.4

Figure 4 - A snapshot of the conformation of the system urease - hydroxamate anion after 2 ns trajectory. Click on the image to retrieve a pdb file.

The agreement with the Stemmler model is satisfactory. The oxime oxygen acts as a bidentate ligand, bridging two metal atoms and forming, by the carbonyl oxygen, a chelate with the first nickel; we did not observe the dehydration of the enzyme proposed by Stemmler⁶.

Conclusions

The results reported here indicate a good qualitative agreement with the experimental data and confirm the ability of the Molecular Dynamics to describe the inhibition of urease and the reaction mechanism. The comparison with the model suggested by various experimental data confirms that our approach reproduces the principal characteristics of the inhibition mechanism and may be used to design more efficient urease inhibitors. We have to point out that, owing to the reduced dimensions of the binding site adopted in our model, these results should be considered as the first stage of a more extensive investigation on the urease inhibition process. Furthe development should consider atoms within a larger radius from the nickel atoms (e.g. 10-15 Å) and an explicit treatment of the solvent.

Acknowledgements

Thanks are due to MURST and CNR for the financial support.

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