Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S ‐glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1‐1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme‐catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar p K a values depended on the structure of the aliphatic chain, and 1‐propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on p K a as 2,2,2‐trifluoroethanol, with a p K a of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1‐propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities. © 2005 Wiley Periodicals, Inc. Biopolymers 79: 292–299, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com