A promiscuous glutathione transferase transformed into a selective thiolester hydrolase.
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 K(M) of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and K(M) 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)/K(M))/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.[1]References
- A promiscuous glutathione transferase transformed into a selective thiolester hydrolase. Hederos, S., Tegler, L., Carlsson, J., Persson, B., Viljanen, J., Broo, K.S. Org. Biomol. Chem. (2006) [Pubmed]
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