Essential active-site lysine of brain glutamate dehydrogenase isoproteins.
Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 mM 2-oxoglutarate with 2 mM NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.
Kim
S W
SW
Department of Biochemistry, College of Medicine, University of Ulsan, Seoul, Songpa-Ku, Korea.
Lee
J
J
Song
M S
MS
Choi
S Y
SY
Cho
S W
SW
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry / Q000502:physiology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D009243:NAD / Q000494:pharmacology; D010455:Peptides / Q000032:analysis; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011732:Pyridoxal Phosphate / Q000032:analysis
D007527:Isoenzymes; D007656:Ketoglutaric Acids; D010455:Peptides; D009243:NAD; D011732:Pyridoxal Phosphate; D005969:Glutamate Dehydrogenase; D008239:Lysine