This work describes how atomistic simulation can provide some insight in the molecular function of enzymes. The atomic structure for arginase supplied by the work of Kanyo et al. 1996 was used to simulate the access and stability of substrate and inhibitors to the enzyme active site. The enzyme, which is highly distributed in living organisms, catalyzes the hydrolysis of arginine to urea and ornithine and it also serves several functions including a role in the regulation of nitric oxide signaling.

The atomic structure of rat liver arginase has many characteristics, among them there is 15 Å deep active-site cleft in each monomer of the homotrimer, with two Mn²⁺ ions at the base of this cleft. Some amino acids in the active site like aspartate 128 and histidine 141 coordinate the metal ions, and some evidences from Cavalli et al. 1994, Perozich et al. 1998 and Carvajal et al. 1999 show that mutation of these residues causes a lowering or loss of catalytic activity of the enzyme. More specifically mutation of histidine 141 into asparagine leaves the enzyme with only 10% of the activity of the native enzyme and it determines a significant increase in the K_m value for arginine. In fact, the studies from Khangulov et al. 1998 point out that the K_m value for wild type arginase is approximately 1.5 mM compared to > 40 mM for the mutant enzyme. On the other hand, binding of the inhibitor lysine, which competes with arginase for the active site, is about 10 times weaker in the Asn141 mutant than in the wild type enzyme. Further information provided by Carvajal et al. 1999 indicates that the mutation of histidine 141 into phenylalanine also results in a loss of activity with a residual activity of about 10%, but in this case there is no change in the value of K_m.

Molecular dynamics is a technique used to simulate the movements of the atoms in a self consistent way. This means that kinetic energy is added to the forces involved between atoms to give enough force to the atoms to overcome local energy barriers. This technique requires enzyme-atomic, substrate and inhibitors structure knowledge and to handle a set of initial conditions like temperature and length of simulations among others. Most of the simulations in practice are done at time steps of 0.001 ps and therefore a very long time of computational simulation is required to obtain average values for some properties of interest. A simulation process is divided in heating, equilibration and cooling steps and once reached the equilibration, images of the systems are recorded and used later to obtain the averages quantities. In this work, we were interested in registering the effects of substrate and inhibitors in the active site of arginase at 300K and at 500K. On the other hand we were also interested in knowing if given a substrate position for the enzyme how are they distorted by alterations of active site residues. Specifically the changes produced by mutating histidine 141 into phenylalanine and histidine 141 into asparagine, both uncharged residues and of quite different shape than histidine. In order to do that, we needed to set up several initial conditions that are described as follows: a) a plausible control position for substrate and inhibitor and b) the enzyme mutants at the closest initial positions than those within the native enzyme system.

For item a) we have carried out a test of MD simulations at 300K and 500K and the end position of the substrate compared. These resulted in a closed related position and to achieve this we have taken as control position the simulation run at 500K. Then it was compared with the resulting structure produced by the optimization of the substrate followed by MD in Bacillus caldovelox crystal structure reported by Bewley et al. 1999 and we found out similar orientations for amine and carboxyl groups. The position of substrate is shown in Figure 1a and distances of the main functional groups were also measured. First attempts to prepare a control position for the substrate, resulted in the substrate and water molecules expulsion from the enzyme and it was necessary to remove the crystallized water molecules that were at the active site, while retaining the water molecule involved in the coordination of the metals ions. It was assumed that the exclusion of these crystallized water molecules from the active site occurs spontaneously in the active enzyme. For item b) mutants of phenylalanine and asparagine 141 were run using Insigth II, a computer program that allow us to replace residues locally, without modification of other residues. Further, energy optimization of 50-150 cycles yield the final structure for all mutants introducing very low changes in the active site. For this work we have used a different tool, namely HyperChem, because of the easy graphical environment to start simulations for students. Afterward using the same tools, mutation aspartate 128 into asparagine was introduced and after energy optimization, a small change in asparagine position was observed.

Then we have located arginine, the substrate for arginase, in the mutant phenylalanine 141 in same place than the one in native enzyme. After optimization a position slightly out of the active site resulted in this mutant enzyme, which is shown in Figure 1b. Next step, with the system previously heated and equilibrated by molecular dynamics characteristics movements were registered for arginine into this enzyme and we found that the substrate maintained its attraction for the active site and repeatedly it moved out and back into the active site, while it maintained its extended structure. However, the substrate never completely returned to the optimized position that it had before the mutation. A much more drastic change was observed in the mutant asparagine 141, the substrate never assumed a fixed position and continuously traveled around these enzyme molecule, this is shown in Figure 1c and Figure 1d. The mutant asparagine 128 had no effect on substrate position as shown in Figure 2a and Figure 2b, in spite of the small change in asparagine position. By the contrary lysine, the inhibitor of the enzyme located in similar way than arginine into the active site of mutants, showed a broad spectrum of different positions into and out of the active site and therefore it was not possible to find a direct relationship with its kinetic data (Figure 3a, Figure 3b and Figure 3c). This inhibitor showed a high mobility but also a great deviation from its average position as shown in Figure 4 .

The conclusion of this work are two. On one side simulations revealed significant differences in the average displacements of the substrate molecule in the active site of wild-type and mutant enzymes. The more drastic differences ocurred in the mutant asparagine 141. Interestingly, these differences correlated well with the kinetic consequences of replacement of histidine 141 by phenylalanine or asparagines. As Khangulov et al. 1998 and Carvajal et al. 1999 evidenced, mutant phenylalanine 141 and asparagines 141 activities were about 10% of wild-type. The simulations indicate a role for histidine 141 in the positioning of the substrate in the active site as suggested by Bewley et al. 1999 and the main interactions involved between substrate and enzyme should be predominantly of electrostatic nature. On the other side, the usefulness of the comparative approach used here and the validity of the conclusion that mutation of histidine 141 by asparagine is significantly more drastic than a change on phenylalanine is reinforced by the observation of nearly the same position for the substrate in wild-type and asparagine 128 enzymes. This agreed with the fact that aspartate 128 interacts with the metal ion and not with the substrate in arginase as described by Kanyo et al. 1996 and Perozich et al. 1998.