The Reaction of Pyruvate with Saccharopine Dehydrogenase

The preceding paper in this journal has reported that pyruvate could be substituted for 2‐oxoglutarate as a substrate of saccharopine dehydrogenase [ɛ‐N‐(l‐glutaryl‐2)‐l‐lysine: NAD oxidoreductase (l‐lysine‐forming)] in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, l‐lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2‐oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [ɛ‐N‐(l‐glutaryl‐2)‐l‐lysine] was found to act as a potent dead‐end inhibitor of the condensation reactions (of lysine and 2‐oxoglutarate, and of lysine and pyruvate) by forming an abort