Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1.
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.
Chern
M K
MK
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 11529, ROC.
Wu
T C
TC
Hsieh
C H
CH
Chou
C C
CC
Liu
L F
LF
Kuan
I C
IC
Yeh
Y H
YH
Hsiao
C D
CD
Tam
M F
MF
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D003903:Deuterium; D004852:Epoxy Compounds; D005973:Glutamine; D005978:Glutathione; D005982:Glutathione Transferase; D009596:Nitrophenols; D010088:Oxidoreductases; D011522:Protons; D011994:Recombinant Proteins; D014364:Tryptophan; D014443:Tyrosine; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane; C023111:epoxidase