Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

Abstract

␥-Glutamyltranspeptidase (GGT) is a heterodimic enzyme that is generated from the precursor protein through posttranslational processing and catalyzes the hydrolysis of ␥-glutamyl bonds in ␥-glutamyl compounds such as glutathione and͞or the transfer of the ␥-glutamyl group to other amino acids and peptides. We have determined the crystal structure of GGT from Escherichia coli K-12 at 1.95 Å resolution. GGT has a stacked ␣␤␤␣ fold comprising the large and small subunits, similar to the folds seen in members of the N-terminal nucleophile hydrolase superfamily. The active site Thr-391, the N-terminal residue of the small subunit, is located in the groove, from which the pocket for ␥-glutamyl moiety binding follows. We have further determined the structure of the ␥-glutamyl

Donor substrate specificity of bovine kidney gamma-glutamyltransferase

“…Plasma cysteine levels of WT and GGT-knock-out mice were 12 μM and 2 μM, respectively (Lieberman et al, 1996), demonstrating that GGT is significant in cysteine recovery by initiating the cleavage of GSH into its constituent amino acids (Lieberman et al, 1996). Serum GGT is "mainly derived from the liver" (Okada et al, 2006). Elevated levels of serum GGT usually indicates hepatic damage, allowing for the use of GGT as a biomarker of liver damage (Okada et al, 2006;Heisterkamp et al, 2008).…”

“…Elevated levels of serum GGT usually indicates hepatic damage, allowing for the use of GGT as a biomarker of liver damage (Okada et al, 2006;Heisterkamp et al, 2008). GGT exhibits an αββα fold (Okada et al, 2006), as shown in Figure 3.…”

“…In the first step of the reaction cycle of GGT, the γ-glutamyl moiety of the donor substrate (GSH) reacts with a hydroxyl group in the active site of the enzyme to generate a tetrahedral acylenzyme intermediate (Tate and Meister, 1974;Wickham et al, 2011). In the case of the E. coli GGT, the catalytic residue has been identified as Thr391, the N-terminal residue of the light subunit (Inoue et al, 2000;Okada et al, 2006). Release of the donor in its free amine form (e.g., cysteinylglycine) from the tetrahedral intermediate produces the covalent acyl-enzyme (γ-glutamyl enzyme) intermediate (Tate and Meister, 1974;Wickham et al, 2011).…”

“…The crystal structures of E. coli GGT in complex with GSH, and E. coli GGT bound to glutamate, were studied to gain insight into substrate recognition (Okada et al 2006). The γ-glutamyl moiety was buried within the core of the enzyme.…”

“…The residues involved in stabilizing the γ-glutamyl moiety of the donor substrate are conserved across E. coli, H.pylori, human, rat and bovine GGTs ( Figure 6) (Okada et al 2006). …”