1C72

TYR115, GLN165 AND TRP209 CONTRIBUTE TO THE 1,2-EPOXY-3-(P-NITROPHENOXY)PROPANE CONJUGATING ACTIVITIES OF GLUTATHIONE S-TRANSFERASE CGSTM1-1

Summary for 1C72

• Entry DOI: 10.2210/pdb1c72/pdb
• Descriptor: PROTEIN (GLUTATHIONE S-TRANSFERASE), 1-HYDROXY-2-S-GLUTATHIONYL-3-PARA-NITROPHENOXY-PROPANE (3 entities in total)
• Functional Keywords: glutathione s-transferase, 1, 2-epoxy-3-(p-nitrophenoxy)propane, epoxidase activity, steady-state kinetics, transferase
• Biological source: Gallus gallus (chicken)
• Cellular location: Cytoplasm: P20136
• Total number of polymer chains: 4
• Total formula weight: 105128.58

Authors

Chern, M.K.,Wu, T.C.,Hsieh, C.H.,Chou, C.C.,Liu, L.F.,Kuan, I.C.,Yeh, Y.H.,Hsiao, C.D.,Tam, M.F. (deposition date: 2000-02-02, release date: 2000-08-30, Last modification date: 2023-08-09)

Primary citation

Chern, M.K.,Wu, T.C.,Hsieh, C.H.,Chou, C.C.,Liu, L.F.,Kuan, I.C.,Yeh, Y.H.,Hsiao, C.D.,Tam, M.F.
Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1.
J.Mol.Biol., 300:1257-1269, 2000

PubMed Abstract: 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 alpha4 helix, Asp161 and Gln165 on the alpha6 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 k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) 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 K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) 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 k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) 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 A 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 beta1 and alpha1 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.

PubMed: 10903867
DOI: 10.1006/jmbi.2000.3904

Experimental method

X-RAY DIFFRACTION (2.8 Å)