Mechanism of saccharopine dehydrogenase: The last enzyme in the lysine biosynthetic pathway in Saccharomyces cerevisiae.

Abstract

A proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD + and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized, and then does not get involved until lysine is formed at end of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen. Collapse of protonated carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the saccharopine formation direction, finite primary deuterium isotope effects were observed for all parameters with the exception of V2/KNADH, consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple isotope effect, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories suggest that proton transfer exists in at least two sequential transition states.

A number of NAD+ analogues, including NADP+, 3-acetylpyridine adenine dinucleotide (3-APAD+), 3-pyridinealdehyde adenine dinucleotide (3-PAAD+), and thio-nicotinamide adenine dinucleotide (thio-NAD+), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate in the opposite direction. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion, and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates, but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD+ suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with 3 carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 in affinity. The keto acid binding pocket is relatively large and flexible, can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid, and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic and the optimal length of the hydrophobic portion of amino acid carbon side chain is 3 or 4 carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.

The uniqueness of the alpha-aminoadipate (AAA) pathway for lysine biosynthesis in fungi makes it a target for the rapid detection and growth control of pathogenic yeasts and molds. Selective inhibition of the enzyme(s) of this pathway by (an) appropriate substrate analog(s) may control or eradicate the growth of fungal pathogens in vivo. Saccharopine dehydrogenase (SDH) catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-ketoglutarate (alpha-Kg) and lysine using NAD+ as an oxidizing agent, the final step in the AAA pathway.

Kinetic data have been measured for SDH from Saccharomyces cerevisiae , suggesting the ordered addition of NAD+ followed by saccharopine in the physiologic reaction direction. In the opposite direction, NADH adds to the enzyme first, followed by random addition of alpha-Kg and lysine. Lysine inhibits the reaction at high concentrations by binding to free enzyme. The alpha-Kg substrate inhibition and double inhibition by NAD+ and alpha-Kg suggest the existence of an abortive E:NAD +alpha-Kg complex. Saccharopine product inhibition suggests a practical irreversibility of the reaction at pH 7.0, in agreement with the overall Keq, and the existences of E:NADH:saccharopine and E:NAD +:saccharopine complexes. Dead-end inhibition studies are consistent with the steady-state random mechanism, and also suggest that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. S-parabolic noncompetitive inhibition of glutarate indicates the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10-7 M, in very good agreement with the Haldane relationship.