A homology model for pig liver esterase was generated on the basis of human carboxyl esterase (hCE) and subjected to extensive molecular dynamics simulations. By virtual mutations the isoenzymes PLE1-6 and APLE were obtained, and the PLE1 trimer was built from the respective model of hCE. Stable structures for all systems were attained after simulations in solution for 12-18 ns, and contact zones between the monomers in the trimer are described. By evaluation of RMSD values of the residues in the monomer a rigid backplane with a number of β-strands and a flexible front part containing several α helices are distinguished. All mutations are concentrated in the soft part, and significant differences in the folding states of the helices were distinguished between the isoenzymes. Substrate access to the active site passes through two helices whose structures are affected by mutations. Variations in substrate specificity between the isoenzymes are ascribed to the structure of the entrance channel rather than to the conformation of the active site itself. The assignment of the residue with a negative side chain stabilizing the histidine protonation in the catalytic triad was revised, being GLU 452 in some isoenzymes rather than GLU 336, which would be the correspondent to most hydrolases. Arguments for this new assignment are given on the basis of simulations and statistics from the 3DM database for hydrolases.