Significance of Arg-107 and Glu-108 in the catalytic mechanism of human gamma-glutamyl transpeptidase. Identification by site-directed mutagenesis.
The catalytic properties of wild-type and mutant enzymes produced by expression of cDNA (for gamma-glutamyl transpeptidase) in COS-1 cells were analyzed. Substitutions of Lys-100 to Asn, Glu-102 to Gln, and ARg-112, -139, -147, and -150 to Gln resulted in expression of fully active enzymes. Replacement of Arg-107 with Gln or His led to complete loss of enzyme activity, whereas substitution to Lys resulted in partial loss of activity, indicating that the positive charge at position 107 is essential for enzyme activity. In the Arg-107-to-Lys mutant, the apparent Km for the donor L-gamma-glutamyl-3-carboxy-4-nitroanilide was increased 8.4-fold compared to that of the wild-type enzyme. Although 32% of the wild-type hydrolysis activity was retained in this mutant, the activity could not be enhanced by addition of an acceptor substrate, glycylglycine, indicating that the transpeptidation reaction was specifically prevented. Substitution of Glu-108 to Gln increased the apparent Km for glycylglycine 8.5-fold while increasing the apparent Km for the donor only 1.6-fold. The apparent Vmax was decreased to 4-5% of wild-type for both substrates. These data suggest that Arg-107 plays a significant role in substrate binding rather than catalysis, and that Glu-108 is an important, although not essential, participant in both acceptor binding and catalysis.
Ikeda
Y
Y
Department of Biochemistry, Osaka University Medical School, Japan.
Fujii
J
J
Taniguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA; D005973:Glutamine / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D004247:DNA; D001120:Arginine; D005723:gamma-Glutamyltransferase; D008239:Lysine