View source for 1c72

[[Image:1c72.gif|left|200px]]<br /><applet load="1c72" size="450" color="white" frame="true" align="right" spinBox="true" 
caption="1c72, resolution 2.8&Aring;" />
'''TYR115, GLN165 AND TRP209 CONTRIBUTE TO THE 1,2-EPOXY-3-(P-NITROPHENOXY)PROPANE CONJUGATING ACTIVITIES OF GLUTATHIONE S-TRANSFERASE CGSTM1-1'''<br />

==Overview==
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.

==About this Structure==
1C72 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Gallus_gallus Gallus gallus] with EPY as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Glutathione_transferase Glutathione transferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.18 2.5.1.18] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1C72 OCA]. 

==Reference==
Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1., Chern MK, Wu TC, Hsieh CH, Chou CC, Liu LF, Kuan IC, Yeh YH, Hsiao CD, Tam MF, J Mol Biol. 2000 Jul 28;300(5):1257-69. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10903867 10903867]
[[Category: Gallus gallus]]
[[Category: Glutathione transferase]]
[[Category: Single protein]]
[[Category: Chern, M.K.]]
[[Category: Chou, C.C.]]
[[Category: Hsiao, C.D.]]
[[Category: Hsieh, C.H.]]
[[Category: Kuan, I.C.]]
[[Category: Liu, L.F.]]
[[Category: Tam, M.F.]]
[[Category: Wu, T.C.]]
[[Category: Yeh, Y.H.]]
[[Category: EPY]]
[[Category: 1]]
[[Category: 2-epoxy-3-(p-nitrophenoxy)propane]]
[[Category: epoxidase activity]]
[[Category: glutathione s-transferase]]
[[Category: steady-state kinetics]]
[[Category: x-ray crystallography]]

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