The Reaction of Pyruvate with Saccharopine Dehydrogenase

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Abstract
A preceding paper reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of [bakers yeast] saccharopine dehydrogenase [EC 1.5.1.7] [.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. A small degree of randomness in the binding of amino and oxo acid substrates may exist. From a product inhibition study, the NAD+ was the last reactant released. Saccharopine [.epsilon.-N-(L-glutaryl-2-L-lysine] acted 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.cntdot.NADH.cntdot.saccharopine complex.

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