A Proposed Proton Shuttle Mechanism for Saccharopine Dehydrogenase from Saccharomyces cerevisiae

Abstract

Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming)) catalyzes the final step in the α-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate α-Kg and lysine using NAD + as an oxidizing agent. 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 pK a of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pK a of 7.2 accepts a proton from H 2 O 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 to give the protonated carbinolamine. Collapse of carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate α-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 reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V 2 /K NADH , consistent with a steady state random mechanism, and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect of D V 2 and D (V 2 /K Lys ), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effect, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V 2 and V 2 /K Lys suggesting proton transfer exists in at least two sequential transition states.Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming); (EC 1.5.1.7)) (SDH 1 ) catalyzes the final step in the α-aminoadipate pathway (AAA) for the de novo synthesis of L-lysine in fungi (1,2). The enzyme catalyzes the reversible † This work is supported by the Grayce B. Kerr Endowment to the University of Oklahoma (to P.F.C.), and a grant (GM 071417) from the National Institute of Health (to P.F.C. and A.H.W.). * Corresponding author: E-mail: pcook@chemdept.chem.ou.edu 1 Abbreviations: SDH, saccharopine dehydrogenase; AAA, α-aminoadipate pathway; α-Kg, α-ketoglutarate; Sacc,...

Determinants of Substrate Specificity for Saccharopine Dehydrogenase from Saccharomyces cerevisiae

“…The 4R-NADD was prepared according to (4). The reaction mixture, contained ethanol-d 6 , NAD, yeast alcohol and aldehyde dehydrogenases, and was titrated to 9.0 with KOH throughout the reaction.…”

“…Data were obtained according to (4). The inhibition constant of AMP was obtained as a function of pH by measuring the initial rate as a function of NADH with α-Kg and lysine fixed at their K m values.…”

“…Since AMP has only one pK a over the pH range 5−10, a value of 6.2−6.4 for the 5'-phosphate, one of the groups with a pK a of 7.0 that must be unprotonated and the group with a pK a of 7.2 that must be protonated must come from the enzyme. On the basis of the proposed chemical mechanism of SDH, the two groups seen in the pK iAMP profile are most likely Asp 281 and Lys 77 (4). The former, with a pK a of about 6.2, is important for binding of saccharopine and likely serves as a general base in the oxidation step, while the group with a pK a of about 7.2, is likely responsible for activating water in hydrolysis of the imine formed upon oxidation of saccharopine, and is known to affect the binding of NAD.…”

“…With the exception of pyridine 2,5-dicarboxylate and L-pipecolic acid, which exhibit no inhibition at high concentrations, and OAA and glutarate, which exhibit noncompetitive inhibition, all other compounds are competitive inhibitor of α-Kg. Glutarate and OAA give noncompetitive inhibition as a result of combination with E-NADH and E-NADH-Lys complexes (4). From these data, Table 6, the keto acid binding site is assessed with respect to the spatial relationship and distance between the C1-C2 unit and the C5 carboxylate.…”

“…The SDH reaction proceeds via an ordered kinetic mechanism in which NAD is the first substrate bound followed by saccharopine in the physiologic reaction direction, while in the reverse reaction direction, NADH adds to enzyme first, and there is no preference for binding of α-Kg and lysine at pH 7.0. A general mechanism for the oxidative deamination of saccharopine has been proposed on the basis of pH-rate profiles and isotope effects, Scheme 1 (4). A group on the enzyme with a pK a of 6.2 is required to deprotonate the secondary amine of saccharopine as it is oxidized to an imine intermediate, and this group ultimately functions to donate a proton to the ε-amine of lysine as it is released as a product.…”