Characterization of Helicobacter pylori γ-Glutamyltranspeptidase Reveals the Molecular Basis for Substrate Specificity and a Critical Role for the Tyrosine 433-Containing Loop in Catalysis^,

Abstract

Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a member of the N-terminal nucleophile hydrolase superfamily. It is translated as an inactive 60 kDa polypeptide precursor that undergoes intramolecular autocatalytic cleavage to generate a fully active heterodimer composed of a 40 kDa and a 20 kDa subunit. The resultant N-terminus, Thr 380, has been shown to be the catalytic nucleophile in both autoprocessing and enzymatic reactions. Once processed, HpGT catalyzes the hydrolysis of the gamma-glutamyl bond in glutathione and its conjugates. To facilitate the determination of physiologically relevant substrates for the enzyme, crystal structures of HpGT in complex with glutamate (1.6 A, Rfactor = 16.7%, Rfree = 19.0%) and an inactive HpGT mutant, T380A, in complex with S-(nitrobenzyl)glutathione (1.55 A, Rfactor = 18.7%, Rfree = 21.8%) have been determined. Residues that comprise the gamma-glutamyl binding site are primarily located in the 20 kDa subunit and make numerous hydrogen bonds with the alpha-amino and alpha-carboxylate groups of the substrate. In contrast, a single hydrogen bond occurs between the T380A mutant and the remainder of the ligand. Lack of specific coordination beyond the gamma-glutamyl moiety may account for the substrate binding permissiveness of the enzyme. Structural analysis was combined with site-directed mutagenesis of residues involved in maintaining the conformation of a loop region that covers the gamma-glutamyl binding site. Results provide evidence that access to this buried site may occur through conformational changes in the Tyr 433-containing loop, as disruption of the intricate hydrogen-bond network responsible for optimal placement of Tyr 433 significantly diminishes catalytic activity.

Donor substrate specificity of bovine kidney gamma-glutamyltransferase

“…In addition, crystal structures of bacterial GGTs, and studies of conserved active site residues of human GGT, indicate that the γ-glutamyl moiety is the primary determinant recognized; the CysGly moiety is believed to be solvent-exposed (Ikeda et al 1993(Ikeda et al , 1995Okada et al 2006;Morrow et al 2007). …”

“…Mammalian and bacterial GGTs contain a donor substrate binding loop that undergoes conformational changes to allow substrate entry into the active site (Okada et al 2006;Morrow et al 2007;Hu et al 2012). …”

“…Morrow and colleagues solved the crystal structure of H. pylori GGT bound to glutamate, and of the catalytically-defective site-directed mutagenesis variant T380A of H. pylori GGT complexed to 4-nitrobenzyl glutathione (NB-GSH) (Morrow et al, 2007). An "extensive hydrogen bond network" for the recognition of the donor substrate was also observed for H.…”

“…In addition, Morrow and colleagues suggested that the Cys-Gly moiety might overlap with the acceptor substrate binding site of H. pylori GGT (Morrow et al, 2007).…”

“…Since substrate is thought to bind deep within the donor substrate binding pocket (consistent with E. coli GGT), the researchers proposed that the substrate binding loop undergoes a conformational change to allow substrate entry into the active site (Morrow et al, 2007). The gating residue (Tyr444) of H. pylori GGT is believed to H-bond to Asn400 (conserved active site residue) and performs a similar function as in E. coli GGT.…”