Add time:09/07/2019         Source:sciencedirect.com

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the α4 helix, Asp161 and Gln165 on the α6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the kcatapp of the enzyme, while a modest 25 % decrease in the kcatapp was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The kcatapp of the S209W mutant is 2.5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The KmGSHapp of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a KmGSHapp and a kcatapp that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in kcatapp was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The KmGSHapp of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested.We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 Å resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting β1 and α1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.

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