Purification and cloning of a gamma-glutamyl transpeptidase from onion (Allium cepa).
Gamma-glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of gamma-glutamyl linkages in gamma-glutamyl peptides and transfer of the gamma-glutamyl group to amino acids and peptides. Although plant gamma-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate gamma-glutamyl-p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion gamma-glutamyl peptide, gamma-glutamyl trans-S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme.
Shaw
Martin L
ML
New Zealand Institute for Crop & Food Research Limited, Germplasm Enhancement, Private Bag 4704, Christchurch 8000, New Zealand.
Pither-Joyce
Meeghan D
MD
McCallum
John A
JA
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019697:Onions / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D021122:Protein Subunits; D005723:gamma-Glutamyltransferase