PMID: 17924658

 

    Legend: Gene, Sites

Title : Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase

Abstract :
  1. Gamma-glutamyltranspeptidase ( GGT ) is a heterodimeric enzyme important for glutathione homeostasis control
  2. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes
  3. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl- enzyme intermediate
  4. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product
  5. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue
  6. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein
  7. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg
  8. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine
  9. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione
  10. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase , and confirmed four of the seven N-linked glycosylation sites
  11. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycosylation, , gamma-glutamyltranspeptidase, -, sites
  • 10. glycosylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
  • 0. gamma-glutamyltranspeptidase, sites
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
53 min-1 966 9
gamma-glutamyltranspeptidase 102724197 0,10
Gamma-glutamyltranspeptidase 102724197 1
min-1 966 8