Mechanisms of enzymes of the alpha-aminoadipate pathway: Homocitrate synthase from Thermus thermophilus and saccharopine dehydrogenase from Saccharomyces cerevisiae

The alpha-aminoadipate (AAA) pathway for lysine biosynthesis is nearly unique to higher fungi, including human and plant pathogens and euglenoids; an exception is the thermophilic bacterium Thermus thermophilus. Knock-out of the genes in this pathway has been shown to be lethal in Saccharomyces cerevisiae. It has been shown that scavenging of lysine is insufficient for survival in the host. Thus, enzymes of this pathway could be potential drug targets. The AAA pathway is comprised of eight enzymatic reactions catalyzed by seven enzymes. Homocitrate synthase (HCS) catalyzes the first and regulated step in this pathway, the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-Kg) to give homocitrate and coenzyme A (CoASH). The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal activated enzyme with either Mg 2+ or Mn2+ capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with alpha-Kg binding prior to AcCoA with Mn2+, while it is steady state random with Mg2+, suggesting a difference in the competence of the E·Mn·alpha·Kg·AcCoA and E·Mg·alpha·Kg·AcCoA complexes.;Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the alpha-aminoadipate pathway, the conversion of L-saccharopine to L-lysine and alpha-ketoglutarate using NAD2+ as an oxidant. The enzyme utilizes a general acid-base mechanism to carry out the multistep saccharopine dehydrogenase reaction with a base proposed to accept a proton from the secondary amine in the oxidation step and a second group proposed to activate water to hydrolyze the imine. A pair of thiols in the dinucleotide binding site forms a disulfide in the wild type (WT) enzyme as isolated, which interferes with binding of the dinucleotide substrate. The SDH enzyme with a C205S mutation, has been characterized recently and is referred to as a pseudo-WT enzyme. Crystal structures of an open apo-form of the pseudo-WT SDH (C205S), as well as a closed form of the C205S enzyme with saccharopine and NADH bound have been solved. The structure of a ternary complex between the C205S pseudo-WT enzyme, NADH, and Sacc provided a closed form of the enzyme and a more accurate description of the interactions between enzyme side chains and reactant functional groups. Importantly, the distance between C4 of the nicotinamide ring to C8 of Sacc is 3.6 A, a reasonable hydride transfer distance. The side chains of H96 and K77 now appear properly positioned to act as acid-base catalysts. Mutation of K77 to M results in a 145-fold decrease in V/Et and greater than a three order of magnitude increase in V2/KLysEt and V2/Kalpha·Kg Et. A primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/K Lys were observed, suggesting that hydride transfer is rate-limiting. The hypothesis was corroborated by the value of 2.0 obtained when the primary deuterium kinetic isotope effect was repeated in D2O. The viscosity effect of 0.8 observed on V2/KLys indicated the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. The deuterium isotope effect on V is slightly lower than that on V/K and decreases when repeated in D2O, suggesting a contribution to rate limitation of product release, likely release of NAD +.;Lysine13, positioned near the active site base (K77), hydrogen-bonds to a glutamate neutralizing it, contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate16 hydrogen-bonds with N-epsilon of R18 which in turn has strong H-bonding interactions with alpha-carboxylate of alpha-Kg. Mutation of K13 to M and E16 to Q decreased kcat ∼ 15-fold, and primary and solvent deuterium isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting of SDH reaction. The pH-rate profiles for K13 exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pK as of catalytic groups. Elimination of E16 affects optimal positioning of R18 for binding and holding alpha-Kg in the correct conformation for optimum catalysis. As a result, the DeltaDeltaG°' value of 2.60 kcal/mol for E16 suggests its contribution in binding of Lys. (Abstract shortened by UMI.).