Identification of Catalytic Nucleophile of Escherichia coli γ-Glutamyltranspeptidase by Mechanism-Based Affinity Label (MOLECULAR BIOFUNCTION-Chemistry of Molecular Biocatalysts)

Abstract

γ-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of -glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a -glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is -glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli -glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a -phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (kon = 4.83 104 M-1sec-1). The inactivation was competitive with respect to the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated the one molecule of small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli -glutamyltranspeptidase. This result strongly suggests that - glutamyltranspeptidase is a new member of N-terminal nucleophile hydrolase family.