Abstract
Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.
Highlights
Be a virulence factor in infection [3, 4]
The Thr380 with alanine (T380A)-Duet protein did not exhibit any catalytic activity with the substrate analog glutamic acid ␥-(4-nitroanilide) (GNA) (Table 2), indicating that Thr380 is required for enzymatic activity
Helicobacter pylori ␥-glutamyltranspeptidase (HpGT) has been implicated in gut colonization and persistence by H. pylori as well as in the progression of diseases resulting from infection by these bacteria [3, 4]
Summary
Generation of Wild-type and Mutant HpGT Expression Constructs—The isolation of HpGT has been described [3], and its sequence corresponds to a predicted open reading frame within the H. pylori genome (KEGG Data Base entry HP1118) [18, 19]. The resultant 0.6-kb PCR product was digested with the relevant enzymes and ligated into a digested pET-24a expression vector (Novagen), incorporating a C-terminal histidine tag. The full-length HpGT expression construct was used as a template, and the HpGT sequence corresponding to the 20-kDa subunit was amplified by PCR, incorporating NdeI and XhoI restriction sites. The resultant 1-kb fragment was gel-purified and ligated into a digested pETDuet expression vector containing the 20-kDa subunit sequence. For HpGT generated using the pETDuet expression construct (HpGT-Duet), the enzyme was isolated in its mature form, and additional incubations were not required. Molecular Mass Determination of HpGT—Purified HpGT was loaded onto a Superdex 200 HR 10/30 gel filtration column (Amersham Biosciences) and separated by fast protein liquid chromatography in 50 mM NaPO4 (pH 7.0) containing 150 mM NaCl at a flow rate of 0.25 ml/min. Electrospray mass spectrometric analyses were performed at the Nebraska Redox Biology Center Metabolomics Core Facility of the University of Nebraska (Lincoln)
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