Identification of a highly reactive threonine residue at the active site of gamma-glutamyl transpeptidase.
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.
Stole
E
E
Department of Biochemistry, Cornell University Medical College, New York, NY 10021.
Seddon
A P
AP
Wellner
D
D
Meister
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007555:Isoxazoles / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D013912:Threonine; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D007555:Isoxazoles; D013912:Threonine; D005723:gamma-Glutamyltransferase; C006303:acivicin