Autocatalytic Processing of γ-Glutamyltranspeptidase

Suzuki, Hideyuki; Kumagai, Hidehiko

doi:10.1074/jbc.m207680200

Cited by 126 publications

(126 citation statements)

References 62 publications

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“…Interestingly, the C terminus of the L subunit and the N terminus of the S subunit are quite distant from one another with Ser-387 C and Thr-391 N being 36 Å apart. The peptide bond between Gln-390 and Thr-391 is cleaved autocatalytically by posttranslational processing (16). Although exact positions of the three C-terminal residues (388-390) are unknown because of disorder of these residues, it is certain that conformational change has occurred upon processing, most probably in the C-terminal segment of the L subunit.…”

Section: Resultsmentioning

confidence: 99%

“…The ability to highly purify large quantities of soluble enzyme makes E. coli GGT an ideal enzyme to study the general reaction mechanisms of this class of enzyme. Using this system, we established that autocatalytic posttranslational processing occurs in the precursor protein, in which Thr-391 acts as the nucleophile, to produce the large (L) and small (S) subunits (16). We also identified that the O␥ atom of N-terminal Thr-391 of the S subunit acts as the nucleophile in its enzymatic reaction (17).…”

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confidence: 96%

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Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

Okada

Suzuki

Wada

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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␥-Glutamyltranspeptidase (GGT) is a heterodimic enzyme that is generated from the precursor protein through posttranslational processing and catalyzes the hydrolysis of ␥-glutamyl bonds in ␥-glutamyl compounds such as glutathione and͞or the transfer of the ␥-glutamyl group to other amino acids and peptides. We have determined the crystal structure of GGT from Escherichia coli K-12 at 1.95 Å resolution. GGT has a stacked ␣␤␤␣ fold comprising the large and small subunits, similar to the folds seen in members of the N-terminal nucleophile hydrolase superfamily. The active site Thr-391, the N-terminal residue of the small subunit, is located in the groove, from which the pocket for ␥-glutamyl moiety binding follows. We have further determined the structure of the ␥-glutamyl

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 96%

Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

Okada

Suzuki

Wada

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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158

211

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“…After autocleavage, T381 forms the N-terminus of the small subunit and serves as the catalytic residue in the active site of hGGT1 (6). This intramolecular autocatalytic maturation is essential to the functional activation of the enzyme and is a hallmark of all known GGT orthologs, ranging from bacteria to humans (3,6,24,46,47). The mature extracellular heterodimer is held together by electrostatic interactions and remains tethered to the plasma membrane by a single-pass transmembrane domain (a.a. that is located at the N-terminus of the large subunit (Fig.…”

mentioning

confidence: 99%

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

West

Wickham

Parks

et al. 2013

Antioxidants & Redox Signaling

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Aims: Human c-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize c-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX 3 C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell.

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“…One unique feature of the Ntnhydrolases is that a single nucleophilic residue is used as the reactive nucleophile for both the autoproteolytic and enzymatic activity (14 -17). The N-terminal nucleophile is Cys in glucosamine-6-phosphate synthase (18) and asparagine synthase (19); Ser in penicillin acylase (20,21); and Thr in glycosylasparaginase (GA) (14,16,22), the proteasome (23), ␥-glutamyltranspeptidase (24), and Taspase1 (25). In addition to these proteolytic enzymes, several other proteins such as hedgehog proteins (26,27), inteins (28), and nucleoporins (29,30) also belong to the Ntn-hydrolase family.…”

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confidence: 99%

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

Lin¹,

Chang²,

Davies³

et al. 2004

Journal of Biological Chemistry

189

187

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Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu2Ser 518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P ؉1 cleavage site (Ser 518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P ؊2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cellfree system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cisproteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.

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Autocatalytic Processing of γ-Glutamyltranspeptidase

Cited by 126 publications

References 62 publications

Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

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