Raman evidence for product binding to the enzyme W137F 4‐chlorobenzoyl‐CoA dehalogenase in two conformational states

The enzyme 4-chlorobenzoyl-CoA dehalogenase catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl-CoA (4-CBA-CoA) to yield 4-hydroxybenzoyl-CoA (4-HBA-CoA). Part of the catalytic mechanism involves interaction between an active site side-chain, from Asp145, with the 4-position of the benzoyl moiety. For the complex consisting of product 4-HBA-CoA in the active site the interaction between Asp145 and the 4-OH group helps produce a major rearrangement in the benzoyl's π-electron system with the system becoming highly polarized. This leads to a large red shift in the benzoyl λmax, from 260 to 370 nm, and radical changes in the Raman spectrum. In addition to the Asp145-4-OH interaction, the aspartate side-chain is held in place by a hydrogen bond to the indole side-chain of Trp137. We explored the consequences of removing this hydrogen bonding interaction by using protein engineering techniques to replace Trp137 by Phe, Ala or His. For 4-HBA-CoA binding to Trp137Phe dehalogenase there is spectroscopic evidence for two conformational populations for bound product. One species (highly polarized) has a λmax near 375 nm and intense Raman bands at 1550, 1520 and 658 cm−1, whereas the second species (moderately polarized) has a λmax near 330 nm and an intense Raman mode at 1575 cm−1. Raman data collected between 6 and 34 °C demonstrate population changes with the highly polarized species having the highest thermodynamic stability. Copyright © 2005 John Wiley & Sons, Ltd.