title: Effects of substitutions of the conserved histidine residues in human gamma-glutamyl transpeptidase.
abstract: gamma-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-gamma-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the Vmax value to 14% of that of the wild type, but had no effect on Vmax/K(m). In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/K(m) for L-gamma-glutamyl-p-nitroanilide as a gamma-glutamyl donor, but with a significant reduction of Vmax/K(m) for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the gamma-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the Vmax value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the Vmax value for the hydrolysis reaction. However, this substitution did not affect Vmax/K(m) for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmax value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the gamma-glutamyl-enzyme through hydrolysis or transfer of the gamma-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the gamma-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue(s) might play an essential role in the enzyme function.
authors.affiliation: Department of Biochemistry, Osaka University Medical School.
authors.forename: Y
authors.initials: Y
authors.lastname: Ikeda
authors.forename: J
authors.initials: J
authors.lastname: Fujii
authors.forename: N
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism; D005973:Glutamine / Q000031:analogs & derivatives / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera / Q000235:genetics; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005973:Glutamine; D006639:Histidine; C001300:gamma-glutamine-4-nitroanilide; D005723:gamma-Glutamyltransferase; D000409:Alanine