caa a22 4500 397556292 CHVBK 20180308164803.0 cr unu---uuuuu 161202e199606 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021363 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021363 Effects of Substitutions of the Conserved Histidine Residues in Human γ-Glutamyl Transpeptidase [Elektronische Daten] [Yoshitaka Ikeda, Junichi Fujii, Naoyuki Taniguchi] γ-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-γ-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/Km. In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/Km for L-γ-glutamyl-p-nitro-anilide as a γ-glutamyl donor, but with a significant reduction of Vmax/Km for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the γ-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/Km for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmttx value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the γ-glutamyl-enzyme through hydrolysis or transfer of the γ-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the γ-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 residues) might play an essential role in the enzyme function. © by the Journal of Biochemistry Regular Papers nationallicence γ-glutamyl transpeptidase nationallicence histidine nationallicence Ikeda Yoshitaka Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut Fujii Junichi Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut Taniguchi Naoyuki Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1166-1170 0021-924X 119:6<1166 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021363 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021363 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ikeda Yoshitaka Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Fujii Junichi Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Taniguchi Naoyuki Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1166-1170 0021-924X 119:6<1166 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford