An approach to optimizing the active site in a glutathione transferase by evolution in vitro

A glutathione transferase (GST) mutant with four active-site substitutions (Phe(10) --> Pro/Ala(12) --> Trp/Leu(107) --> Phe/Leu(108) --> Arg) (C36) w as isolated from a library of active-site mutants of human GST Al-1 by the combination of phage display and mechanism-based affinity adsorption [Hanss on, Widersten and Mannervik (1997) Biochemistry 36, 11252-11260]. C36 was s elected on the basis of its affinity for the transition-state analogue 1-(S -glutathionyl)-2,4,6-trinitrocyclohexadienate. C36 affords a 10(5)-fold rat e enhancement over the uncatalysed reaction between reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB), as evidenced by the ratio between k(ca t)/K-m and the second-order rate constant k(2). The present study shows tha t C36 can evolve to an even higher catalytic efficiency by an additional si te-specific mutation. Random mutations of the fifth active-site residue 208 allowed the identification of Is variants, of which the mutant C36 Met(208 ) --> Cys proved to be the most active form. The altered activity was subst rate selective such that the catalytic efficiency with CDNB and with 1-chlo ro-6-trifluoromethyl-2,4-dinitrobenzene were increased 2-3-fold, whereas th e activity with ethacrynic acid was decreased by a factor of 8. The results show that a single-point mutation in the active site of an enzyme may modu late the catalytic activity without being directly involved as a functional group in the enzymic mechanism. Such limited modifications are relevant bo th to the natural evolution and the in vitro redesign of proteins for novel functions.