Identification of a highly reactive threonine residue at the active site of gamma-glutamyl transpeptidase.

Abstract

gamma-Glutamyl transpeptidase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2], an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-[3-14C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme.