The reaction of pyruvate with saccharopine dehydrogenase.
The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-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 [epsilon-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 abortive E. NADH. saccharopine complex.
D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011773:Pyruvates; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012443:Saccharopine Dehydrogenases / Q000378:metabolism*
D011773:Pyruvates; D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases; D008239:Lysine