Substrate binding water expulsion

Substrate binding and activation are followed by attack of the carboxylate side chain of Asp-145 at the benzoyl C-4 atom to give an enzyme-stabilized Meisenheimer intermediate (EMc) (Figure 8). Indeed, a site-directed mutant in which Asp-145 has been replaced by an alanine is catalytically inactive." Ketonization of the EMc results in rearomatization of the benzoyl ring and expulsion of the chloride. This nucleophilic addition-elimination mechanism (SNAr-type reaction) results in a second covalent (aryl-enzyme) intermediate, which is subsequently hydrolyzed by a water molecule that is activated by His-90 to give the free enzyme and the product. The existence of a covalent aryl-enzyme intermediate has been inferred from 0-labeling studies (similar to those described for haloalkane and haloalcohol dehalogenase) and from the direct measurement of the aryl-enzyme... 

Water expulsion can, however, supply a thermodynamic driving force for substrate binding in some large hydrophobic cavities, from which water expulsion... 

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