Active-site amino acid residues in gamma-glutamyltransferase and the nature of the gamma-glutamyl-enzyme bond.


Active-site residues in rat kidney gamma-glutamyltransferase (EC were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by beta-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the gamma-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[(14)C]acetamide. One of these possessed a carboxy group, which formed a [(14)C]glycollamide ester, and the other was cysteine, shown by isolation of S-[(14)C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[(14)C]acetamide. 4. Isolation of the gamma-[(14)C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The gamma-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the gamma-[(14)C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the gamma-[(14)C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with gamma-glutamyl-epsilon-lysine. 5. A scheme for the catalytic mechanism is proposed.

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@article{Elce1980ActivesiteAA, title={Active-site amino acid residues in gamma-glutamyltransferase and the nature of the gamma-glutamyl-enzyme bond.}, author={John S. Elce}, journal={The Biochemical journal}, year={1980}, volume={185 2}, pages={473-81} }