MODIFICATION OF AN ESSENTIAL AMINO GROUP OF GLUTATHIONE-REDUCTASE FROM YEAST BY PYRIDOXAL 5'-PHOSPHATE

Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate ( PLP). The reactivation of the enzyme by dilution as well as a characte ristic absorption peak at 325 nm exhibited by NaBH4 - reduced - PLP mo dified enzyme show that the inactivation is due to the specific modifi cation of the epsilon-amino group of lysine residue. The maximum of 70 % inactivation was observed at 7 mM PLP and the equilibrium was reache d within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent inte rmediate is formed prior to inactivation. From the studies on the effe ct of pH on the inactivation rate, the pKa of epsilon-amino group of t he reactive lysine residue was calculated to be 7.3. Among Various pro tecting agents tried, only NADP was found to be effective. The apparen t 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 PL P concentration.