title: Modification of an essential amino group of glutathione reductase from yeast by pyridoxal 5'-phosphate.
abstract: Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP). The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4-reduced-PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue. The maximum of 70% inactivation was observed at 7mM PLP and the equilibrium was reached within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation. From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3. Among various protecting agents tried, only NADP was found to be effective. The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kanpur, India.
authors.forename: A
authors.initials: A
authors.lastname: Pandey
authors.forename: L
authors.initials: L
authors.lastname: Iyengar
authors.forename: S S
authors.initials: SS
authors.lastname: Katiyar
mesh_terms: D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019803:Glutathione Disulfide / Q000378:metabolism; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D009249:NADP; D011732:Pyridoxal Phosphate; D005980:Glutathione Reductase; D008239:Lysine; D019803:Glutathione Disulfide