Redesign of substrate-selectivity determining modules of glutathione transferase A1-1 installs high catalytic efficiency with toxic alkenal products of lipid peroxidation

Abstract

The evolution of proteins for novel functions involves point mutations and recombinations of domains or structural segments. Mimicking this process by rational design in vitro is still a major challenge. The present report demonstrates that the active site of the enzyme glutathione transferase (GST) A1-1 can be tailored for high catalytic efficiency with alkenals. The result is a >3,000-fold change in substrate selectivity involving a noteworthy change in preferred catalyzed reaction from aromatic nucleophilic substitution to Michael addition. The hydrophobic substrate binding pocket of GST A1-1 is formed by three structural modules, which were redesigned sequentially with four point mutations and the exchange of a helical segment. The substitutions were made to mimic first-sphere interactions with a substrate in GST A4-4, which naturally has high activity with alkenals. These substrates are toxic lipid peroxidation products of pathophysiological significance, and glutathione conjugation is a route of their inactivation. The final product of the sequential redesign of GST A1-1, mutant GIMFhelix, had a 300-fold increase in catalytic efficiency with nonenal and a >10 times decreased activity with 1-chloro-2,4-dinitrobenzene. In absolute values, GIMFhelix is more efficient than wild-type GST A4-4 with some alkenal substrates, with a k(cat)/K(m) value of 1.5 +/- 0. 1 10(6) M(-1) small middle dots(-1) for nonenal. The pKa value of the active-site Tyr-9 of GIMFhelix is 7.3 +/- 0.1, approaching the unusually low value of GST A4-4. Thus, rational redesign of the active-site region of an enzyme may be sufficient for the generation of efficient catalysts with altered chemical mechanism and novel selectivity.

Figure 1

The three hydrophobic substrate-binding modules of human GST A1–1 (2) and human GST A4–4 (17): the β1-α1 loop (residues 9–16), the C-terminal part of the α4 helix (residues 104–111), and the C terminus of the protein (residues 208–222, where 210–220 form the α9 helix). GST A4–4 residues considered important for proper first-sphere interactions with alkenal substrates and installed into corresponding positions in GST A1–1 are boxed. Residue numbers are indicated above the residues at each end.

Figure 2

Two distinct chemical reactions efficiently catalyzed by GST A1–1 and GST A4–4. The reaction mechanisms differ; GST A1–1 favors an aromatic substitution reaction by which the sulfur of glutathione (γ-Glu-Cys-Gly) replaces the chlorine atom of CDNB. GST A4–4 has the highest activity in the Michael addition reaction where glutathione is added to the activated double bond of an alkenal. One subunit of each enzyme is shown (A1 and A4).

Figure 3

Close view of the active site of GST A1–1 (2). The ligand, S-benzylglutathione, which defines the position of the active site, is colored red. The following mutations have been introduced in mutant GIMFhelix: Ala-12–Gly in the β1-α1 loop (blue) and Leu-107–Ile, Leu-108–Met, and Val-111–Phe in the C-terminal part of α4-helix (yellow). The C terminus (green) of GST A1–1 has been replaced with the corresponding residues of GST A4–4. This substitution altered 10 amino acid residues and included the mutations Ser-212–Tyr and Met-208–Pro, believed to be important for the change of the mechanism to a Michael addition.

Figure 4

Substrate selectivity profiles expressed as catalytic efficiency, k_cat/K_m, of mutants Ghelix and GIMFhelix as well as of wild-type GST A1–1 and GST A4–4, with the following substrates: hexenal (black), hydroxyhexenal, HHE (gray), nonenal (hatched), and HNE (white). The k_cat/K_m values for GST A4–4 obtained with HHE and HNE were determined previously (13). The kinetic parameters for hexenal and nonenal are from Table 2, and for HHE and HNE as follows: GST A1–1, K_m^HNE = 0.047 ± 0.002 mM, k_cat^HNE = 2.4 ± 0.05 s^-1; Ghelix, K_m^HNE = 0.047 ± 0.017 mM, k_cat^HNE = 6.3 ± 1.3 s^-1; GIMFhelix, K_m^HHE = 0.39 ± 0.07 mM, k_cat^HHE = 42.9 ± 6.4 s^-1, K_m^HNE = 0.011 ± 0.003 mM, k_cat^HNE = 11.6 ± 0.7 s^-1; and GST A4–4 (13), K_m^HHE = 0.23 ± 0.05 mM, k_cat^HHE = 23 ± 3 s^-1, K_m^HNE = 0.037 ± 0.004 mM, k_cat^HNE = 113 ± 4 s^-1.