Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde.
Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.
Ahn
J Y
JY
Department of Biochemistry, University of Ulsan College of Medicine, Seoul, South Korea.
Choi
S
S
Cho
S W
SW
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D020134:Catalytic Domain; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009764:o-Phthalaldehyde; D005969:Glutamate Dehydrogenase; D008239:Lysine