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

被引:87
作者
Inoue, M
Hiratake, J [1 ]
Suzuki, H
Kumagai, H
Sakata, K
机构
[1] Kyoto Univ, Chem Res Inst, Uji, Kyoto 6110011, Japan
[2] Kyoto Univ, Grad Sch Biostudies, Sakyo Ku, Kyoto 6068502, Japan
关键词
D O I
10.1021/bi000220p
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 timedependent 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 lable by phosphonylating the putative catalytic 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.
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页码:7764 / 7771
页数:8
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