Role of an electrostatic network of residues in the enzymatic action of the Rhizomucor miehei lipase family.

Abstract

We have used continuum electrostatic methods to investigate the role of electrostatic interactions in the structure, function, and pH-dependent stability of the fungal Rhizomucor miehei lipase (RmL) family. We identify a functionally important electrostatic network which includes residues S144, D203, H257, Y260, H143, Y28, R80, and D91 (residue numbering is from RmL). This network consists of residues belonging to the catalytic triad (S144, D203, H257), residues located in proximity to the active site (Y260), residues stabilizing the geometry of the active site (Y28, H143), and residues located in the lid (D91) or close to the first hinge (R80). The lid and the first hinge are associated with the interfacial activation of lipases, where an alpha-helical lid opens up by rotating around two hinge regions. All network residues are well conserved in a set of 12 lipase homologues, and 6 of the network residues are located in sequence motifs. We observe that the effects of modeled mutations R86L, D91N, and H257F on the pH-dependent electrostatic free energies differ significantly in the closed and open conformations of RmL. Mutation R86L is especially interesting since it stabilizes the closed conformation but destabilizes the open one. Site-site electrostatic interaction energies reveal that interactions between R86 and D61, D113, and E117 stabilize the open conformation.