The reaction of pyridoxal 5'-phosphate with an essential lysine residue of saccharopine dehydrogenase (L-lysine-forming).

Saccharopine dehydrogenase (EC 1.5.1.7) from bakers’ yeast was reversibly inactivated by pyridoxal and pyridoxal 5’-phosphate. The inactivation by pyridoxal 5’-phosphate was accompanied by the appearance of a peak at 428 nm, which was shifted to 325 nm upon reduction with NaBH4. Paper chromatography of an acid hydrolysate of the pyridoxal 5’-phosphate-treated, NaBH4-reduced enzyme showed the presence of e N pyridoxyllysine, indicating that the inactivation was due to Schiff base formation with a lysyl residue of the enzyme. Correlation between the loss of enzyme activity and the amount of pyridoxal 5’-phosphate incorporated into the enzyme showed that the modification of 1 lysyl residue per molecule of enzyme completely inactivated the enzyme. The inactivation was fully protected by a-ketoglutarate in the presence of a saturating concentration of NADH. Since a-ketoglutarate is bound only to the enzymeoNADH complex, the result strongly suggests that the lysine residue essential for activity is located in the vicinity of the substrate-binding site. The kinetics of inactivation by pyridoxal 5’phosphate and reactivation of the inactivated enzyme, as well as the relationship between the residual activity at equilibrium and pyridoxal 5’-phosphate concentration, indicated that no noncovalent enzyme pyridoxal 5’-phosphate complex was formed prior to Schiff base formation, in contrast to many enzymes that have organophosphates as substrates. From the variation of the equilibrium constant for inactivation with pH, a pK value of about 8.8 was obtained for the lysyl residue.