Structural Instability of the Active Site of T1 Lipase Induced by Replacement of Na(+) with Water Complexed with the Phenylalanine Aromatic Ring.

Abstract

The crystallographic analysis of T1 lipase suggested an interaction between Na(+) and the aromatic ring of Phe16 in the active site. However, experimental approaches could not dismiss the possible presence of water instead of Na(+). Our previous molecular dynamics (MD) simulations suggested that the significantly large enthalpy gain of the Na(+)-π interaction was required to preserve the catalytic core structure of T1 lipase. In this study, to examine the effects of water, we performed further MD simulations of T1 lipase involving the water-π interaction, instead of the Na(+)-π interaction, exploiting various force fields, such as ff99, ff02, and an accurate potential field to describe the water-π interaction, which was generated using our recently developed scheme (referred to as the grid-based energy representation). The analyses revealed that the water-π complex was unstable in the catalytic core of T1 lipase even when the accurate potential of the water-π complex represented by the grid-based energy function was employed in the MD simulations and led to the disruption of the coordinated structure. In contrast, the catalytic core structure of T1 lipase involving the Na(+)-π complex was significantly stable in the 10 ns MD simulation using the grid-based energy representation of the Na(+)-π interaction. Thus, the possible presence of water may be excluded, and our previous proposal concerning the functional role of the structural element involving the Na(+)-π interaction in the catalytic site of T1 lipase has unambiguously been confirmed. Further, the strong coordination of Na(+) and Nε of His358 was also shown to be substantial to preserve the core structure of the catalytic site.