PMID:10869181
| Citation |
Inoue, M, Hiratake, J, Suzuki, H, Kumagai, H and Sakata, K (2000) 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. Biochemistry 39:7764-71 |
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| Abstract |
gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione 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 hitherto unknown catalytic nucleophile is gamma-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 gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of 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 one molecule of the 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 gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family. |
| Links | |
| Keywords |
Aminobutyrates/pharmacology; Catalysis; Enzyme Inhibitors/pharmacology; Escherichia coli/chemistry; Escherichia coli/enzymology; Glutamic Acid/analogs & derivatives; Glutamic Acid/chemistry; Glutamic Acid/metabolism; Mass Spectrometry; Threonine/chemistry; Threonine/metabolism; gamma-Glutamyltransferase/antagonists & inhibitors; gamma-Glutamyltransferase/chemistry; gamma-Glutamyltransferase/metabolism |
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