Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?

Abstract

We report the site-specific incorporation of a thiocyanate vibrational probe into the active-site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analogue shifts the observed −CN vibrational frequency by +0.4 cm-1 relative to unliganded KSI, binding of an intermediate steroid analogue containing localized negative charge results in a +2.8 cm-1 shift. On the basis of a Stark tuning rate of 0.7 cm-1/(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the −CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analogue but lacking distal hydrocarbon rings results in an identical −CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibl...