Raman difference spectroscopic studies of dithiobenzoyl substrate and product analogs binding to the enzyme dehalogenase: pi-electron polarization isprevented by the C=O to C=S substitution

The enzyme 4-chlorobenzoyl coenzyme A (CoA) dehalogenase catalyzes the tran sformation of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. High-quality Ram an spectra of substrate or product or their analogs in the enzyme's active site can be obtained using Raman difference spectroscopy. Here, data are pr esented for the substrate and product analog, 4-chlorobenzoyldithio-CoA and 4-hydroxybenzoyldithio-CoA, respectively, where the benzoyl C=O group has been replaced by C=S. The rationale behind this substitution is to explore the consequences of perturbing enzyme active site-ligand C=O interactions. Interpretation of the Raman data for the complexes was placed on a firm foo ting by undertaking density functional theory calculations on the model com pounds S-ethyl 4-chlorobenzoate dithioester and S-ethyl 4-hydroxybenzoate d ithioester. Based on the calculations and on data for the CoA moiety, essen tially all the Raman bands in the spectra of the substrate and product dith io analogs could be assigned with confidence. The calculations show that th e dithioester phenyl moiety is non-planar with the C=S bond typically being twisted 30 degrees out of the plane of the phenyl ring. The Raman differen ce data for the dehalogenase bound dithio-based substrate and product show that only minor spectral changes occur upon binding and this indicates that the conformation about the dithioester bonds is essentially unchanged by b inding. The results for the product analog are in strong contrast to those for the complex involving the natural product, 4-hydroxybenzoyl-CoA, where binding brings about a complete rearrangement of the benzoyl's normal modes . The reorganization is due to a marked polarization of the benzoyl's K-ele ctrons, which, in turn, is brought about by electron pulling fortes acting at the carbonyl and electron pushing forces near the benzoyl 4-position in the active site. However, pi-electron polarization, and the accompanying no rmal mode rearrangement, do not happen for the dithio analog because the C= S substituted group does not transmit the electron pulling forces, that act on the C=O in the natural substrate, to the phenyl ring. The spectroscopic results explain why the dithio-based substrate reacts with dehalogenase 20 0 times more slowly than the thiolester substrate. For the latter, electron pull at the C=O is used to deplete electron density at the benzoyl's 4-pos ition and to facilitate the substitution of the 4-Cl atom by the 4-OH group . Copyright (C) 2000 John Wiley & Sons, Ltd.