Overall Kinetic Mechanism of Saccharopine Dehydrogenase from Saccharomyces cerevisiae^†

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of α-ketoglutarate (α-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by α-Kg and double inhibition by NAD and α-Kg suggest the existence of an abortive E:NAD:α-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K_eq. Saccharopine is noncompetitive versus lysine or α-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and α-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of α-Kg and lysine. Leucine and oxalylglycine serve as lysine and α-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against α-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the α-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)₂ complex as a result of occupying both the lysine- and α-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and α-Kg, suggesting the combination to the E:NADH:α-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 × 10^-7 M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.