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.
Ikeda
Y
Y
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
Fujii
J
J
Taniguchi
N
N
Meister
A
A
D001224:Aspartic Acid / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; C001300:gamma-glutamine-4-nitroanilide; D005723:gamma-Glutamyltransferase; D003545:Cysteine