A promiscuous glutathione transferase transformed into a selective thiolester hydrolase.

ABSTRACT

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.