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

Abstract

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 Mg2+ 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. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d3) in the presence of Mg2+ is unity at low concentrations of AcCoA, while it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg2+ or Mn2+ as the divalent metal ion, while V/Ka-Kg (with Mn2+) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as &alpha-Kg. Lysine binding exhibits negative cooperativity, indicating crosstalk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.

Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the &alpha-aminoadipate pathway, the conversion of L-saccharopine to L-lysine and &alpha-ketoglutarate using NAD+ 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 Å, 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/K&alpha-KgEt. A primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/KLys 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+. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/KLysEt is pH independent consistent with the loss of an acid-base catalyst and perturbation of the pKa of the second catalytic group to higher pH, likely a result of a change in the overall charge in the active site. The H96Q mutation results in about a 28-fold decrease in V2/Et and >103-fold decreases in the second order rate constant. The primary deuterium kinetic isotope effect is within error 1, but a large solvent deuterium isotope effect of 2.4 is observed, suggesting rate limiting imine hydrolysis, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. In agreement, the multiple isotope effects, repeating the primary deuterium effect in D2O and the solvent effect with NADD, are identical to the individual effect. The pH-rate profile for V2/KLysEt exhibits the pKa for K77, perturbed to a value of about 9, which must be unprotonated in order to accept a proton from the epsilon-amine of the substrate Lys so that it can act as a nucleophile. The proposed roles of H96 and K77 are corroborated by the nearly 700-fold decrease in V2/Et and >105-fold decreases in the second order rate constants for the double mutant. Thus, data consistent with an acid-base mechanism in the non-physiologic reaction direction suggest that the K77 side chain initially accepts a proton from the epsilon-amine of the substrate lysine and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and then H96 protonates the carbonyl oxygen as the imine is formed.

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 pKas 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 delta delta G°' value of 2.60 kcal/mol for E16 suggests its contribution in binding of Lys.

Overall, data are consistent with the proposed acid-base mechanism in the non-physiologic reaction direction in which the K77 side chain initially accepts a proton from the &alpha-amine of the substrate lysine and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and then H96 protonates the carbonyl oxygen as the imine is formed. Lysine13 and E16 play an important role of balancing the charge in the active site and elimination of either of the residues perturbs pKas of the catalytic residues, positioning of R18 residue for optimum binding of &alpha-Kg is affected and the step contributing to the rate-limitation is changed to hydride transfer while in the case of C205S enzyme neither hydride transfer nor imine hydrolysis was solely responsible for rate-limitation. Thus K13 and E16 are important for favorable binding of Lys and &alpha-Kg and optimum catalysis.