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

Abstract

AbstractThe 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.