Characterization of Helicobacter pylori gamma-glutamyltranspeptidase reveals the molecular basis for substrate specificity and a critical role for the tyrosine 433-containing loop in catalysis.

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.