title: Involvement of Ser-451 and Ser-452 in the catalysis of human gamma-glutamyl transpeptidase.
abstract: The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.
authors.affiliation: Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Ikeda
authors.forename: J
authors.initials: J
authors.lastname: Fujii
authors.forename: M E
authors.initials: ME
authors.lastname: Anderson
authors.forename: N
authors.initials: N
authors.lastname: Taniguchi
authors.forename: A
authors.initials: A
authors.lastname: Meister
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001881:Borates; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000494:pharmacology; D013552:Swine; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001881:Borates; D017931:DNA Primers; D007555:Isoxazoles; D012694:Serine; D005723:gamma-Glutamyltransferase; C006303:acivicin