Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

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The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Glutathione transferases (GSTs) catalyze the nucleophilic attack of the thiolate of glutathione on a variety of noxious, often hydrophobic, electrophiles. The interactions responsible for the binding of glutathione have been deduced in great detail from the 3-dimensional structures that have been solved for three different GSTs, each a member of a distinct structural class. However, the interactions of the electrophilic substrates with these enzymes are still largely unexplored. The contribution of the active-site Met208 to aromatic and benzylic chloride substitution reactions catalyzed by human class Alpha GST A1-1 has been evaluated by comparison of wild-type enzyme with variants mutated in position 208. The results show that the amino acid residue at position 208 primarily affects the aromatic substitution reaction, tested with 1-chloro-2,4-dinitrobenzene as substrate, possibly by interacting with the delocalized negative charge of the substituted ring structure in the transition state.