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 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.
Inoue
M
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hiratake
J
J
Suzuki
H
H
Kumagai
H
H
Sakata
K
K
D000613:Aminobutyrates / Q000494:pharmacology; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
C411727:2-amino-4-fluorophosphonobutanoic acid; D000613:Aminobutyrates; D004791:Enzyme Inhibitors; D013912:Threonine; D018698:Glutamic Acid; D005723:gamma-Glutamyltransferase