Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal Thr-391 in small subunit is the nucleophile

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutath ione metabolism and catalyzes the hydrolysis and transpeptidation of gamma- glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hither to unknown catalytic nucleophile is gamma-glutamylated. Neither affinity la beling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the iden tification of the catalytic nucleophile of Escherichia coli gamma-glutamylt ranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4 -(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride d erivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a timedependent manner (k(on) = 4.83 x 10(4) M-1 s(-1)). The inactivation rat e was decreased by increasing the concentration of the substrate. The inact ivated enzyme did not regain its activity after prolonged dialysis, suggest ing that 1 served as an active-site-directed affinity lable by phosphonylat ing the putative catalytic Ion-spray mass spectrometric analyses revealed t hat one molecule of 1 phosphonylated one molecule of the small subunit. LC/ MS experiments of the proteolytic digests of the phosphonylated small subun it identified the N-terminal peptide Thr391-Lys399 as the phosphonylation s ite. Subsequent MS/MS experiments of this peptide revealed that the phospho nylated residue was Thr-391, the N-terminal residue of the small subunit. W e conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. c oli gamma-glutamyltranspeptidase. This result strongly suggests that gamma- glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrol ase family.