The Three-dimensional Structure of Cys-47-modified Mouse Liver Glutathione S-Transferase P1-1

Abstract

The three-dimensional structure of mouse liver glutathione S-transferase P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitrobenzyl)glutathione have been determined by x-ray diffraction analysis. The structure of the modified enzyme described here is the first structural report for a P i class glutathione S-transferase with no glutathione, glutathione S-conjugate, or inhibitor bound. It shows that part of the active site area, which includes helix ␣B and helix 3 10 B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged. The position of the sulfur atom of glutathione is occupied in the ligand-free enzyme by a water molecule that is at H-bond distance from Tyr-7. We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the proton from the thiol group of glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the carboxymethylated enzyme results in a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly reduces the affinity. A detailed kinetic study of the modified enzyme indicates that the carboxymethylation increases the K m for glutathione by 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.

Structure of the Major Cytosolic Glutathione S-Transferase from the Parasitic Nematode Onchocerca volvulus

“…Tyr 7 does not appear to play a role as a general base in the enzymatic reaction. Similar conclusions have been reported for other mammalian -class GSTs (19,21,59). …”

The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

“…B zone is stabilized upon binding of the substrates, as observed in the structure of the unliganded Cys-47-carboxymethylated mGST P1-1 enzyme, where the absolutely disordered helices αB and 3 "! B are partially organized upon binding of S-(p-nitrobenzyl)glutathione [30]. We observe also that the binding of a hydrophobic substrate in the form of a GSH adduct [12] increases the definition of the electron density, and decreases the atomic B-factors (although they are still higher than the rest of the protein), in comparison with mGST P1-1:GSH.…”

“…ez and co-workers [12] suggested that, should Tyr-7 be in its ionized form, a positively charged residue could be in closer proximity in the free enzyme and be removed when GSH binds. The structure of the unliganded Cys-47-modified enzyme [30] proves that this is not the case (see below). Other observations [9,31,32] also argue against the tyrosinate hypothesis, and other functional groups of the protein [33] or even the carboxyl groups of GSH [34] have been proposed as the proton acceptors.…”

“…The recent unliganded crystal structures of the human GST class Alpha [17] and the S. japonica GST [23] show an intact G subsite in the absence of substrate or inhibitor. The unliganded Cys-47-modified class-Pi mouse liver GST (mGST P1-1) [30] also shows an unmodified Tyr-7 environment, although some of the GSH-binding residues are disordered. Based on that structure and the preliminary report of the mGST P1-1 in complex with GSH [36], we have proposed a mechanism for thiol activation where a water molecule initially accepts the thiol proton.…”

Characterizing protein modifications by reactive metabolites using magnetic bead bioreactors and LC-MS/MS

“…It is easily expressed in E. coli and well characterized. 14, 15 This relatively small protein features four cysteine residues (Cys-14, 47, 101 and 169), among which Cys-47 has the highest reactivity due to its relatively low pKa (3.5–4.2) and accessibility. 16 …”