Human gamma-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit.

Mutant human gamma-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the alpha-amino group of the gamma-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the gamma-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates gamma-glutamyl enzyme formation.