Tyr115, Gln165 and Trp209 contribute to the 1,2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-11

Add time:07/30/2019    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.

We also recommend Trading Suppliers and Manufacturers of 3-(4-nitrophenoxy)-2-(S-glutathionyl)-1-propanol (cas 134564-85-5). Pls Click Website Link as below: cas 134564-85-5 suppliers

Prev:[11C]Befloxatone (cas 134564-82-2) brain kinetics is not influenced by Bcrp function at the blood–brain barrier: A PET study using Bcrp TGEM knockout rats
Next:The Three-Dimensional Structure of an Avian Class-mu Glutathione S-transferase, cGSTM1-1 at 1.94 Å Resolution1)