Abstract
gamma-Glutamyltranspeptidase is the key enzyme in glutathione metabolism, and we previously presented evidence suggesting that it belongs to the N-terminal nucleophile hydrolase superfamily. Enzymatically active gamma-glutamyltranspeptidase, which consists of one large subunit and one small subunit, is generated from an inactive common precursor through post-translational proteolytic processing. The processing mechanism for gamma-glutamyltranspeptidase of Escherichia coli K-12 has been analyzed by means of in vitro studies using purified precursors. Here we show that the processing of a precursor of gamma-glutamyltranspeptidase is an intramolecular autocatalytic event and that the catalytic nucleophile for the processing reaction is the oxygen atom of the side chain of Thr-391 (N-terminal residue of the small (beta) subunit), which is also the nucleophile for the enzymatic reaction.
Highlights
␥-Glutamyltranspeptidase (GGT1; EC 2.3.2.2), which consists of one large subunit and one small subunit, is the key enzyme in glutathione metabolism and is widely distributed in living organisms [1,2,3,4]
The 100,000 protein was assigned as pro-MBP-GGT and was autocatalytically processed into the 80,000 protein and the 20,000 protein
Autocatalytic Processing Is an Intramolecular Reaction and Not an Intermolecular One—If enzymatically active MBP-GGT and/or its processable precursor, pro-MBP-GGT, could cleave a processing-deficient precursor, e.g. pro-T391A, intermolecularly in vivo, a strain that coexpresses pro-T391A and MBPGGT should give a band corresponding to the molecular weight of the large subunit of the wild-type GGT on Western blot analysis after SDS-PAGE
Summary
The residues of E. coli GGT are numbered as a single chain (residues 1–580) comprising the signal peptide (residues 1–25), the large (␣) subunit (residue 26 –390), and the small () subunit (residues 391–580). T391S means a mutant GGT (protein) whose Thr-391 was replaced by a Ser residue
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