Kinetic studies of saccharopine reductase from Saccharomyces cerevisiae

Saccharopine reductase (SR) (EC 1.5.1.10) is the penultimate enzyme in the alpha-aminoadipate pathway encoded by the LYS9 gene in Saccharomyces cerevisiae. Saccharopine reductase (SR) catalyzes the condensation of L-alpha-aminoadipate-delta-semialdehyde (AASA) with L-glutamate to form an imine which is subsequently reduced by NADPH to give saccharopine. Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of NADPH to the free enzyme followed by AASA, which adds in rapid equilibrium prior to L-glutamate in the forward reaction direction. In the reverse reaction direction, NADP adds to enzyme prior to saccharopine. Product inhibition by NADP is competitive vs. NADPH and noncompetitive vs. AASA and L-glutamate consistent with addition of the dinucleotide prior to the aldehyde; saccharopine is noncompetitive vs. NADPH, AASA and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs. NADP and noncompetitive vs. saccharopine, L-glutamate is noncompetitive vs. both NADP and saccharopine, while AASA is noncompetitive vs. saccharopine and uncompetitive vs. NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogs of AASA, L-glutamate, and saccharopine. 2-amino-6-heptenoic acid was chosen as a dead-end analog of AASA and is competitive vs. AASA, uncompetitive vs. NADPH, and noncompetitive vs. L-glutamate. Ketoglutarate (alpha-Kg) served as a dead-end analog of L-glutamate and is competitive vs. L-glutamate and uncompetitive vs. L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid and L-ornithine were used as dead-end analogs of saccharopine and showed competitive inhibition vs. saccharopine and uncompetitive inhibition vs. NADP.;An acid-base chemical mechanism has been proposed for SR on the basis of pH rate profiles and solvent deuterium kinetic isotope effects. A finite solvent isotope effect is observed indicating that proton(s) are in flight in the rate limiting step(s) and likely the same step is limiting under both limiting and saturating substrate concentrations. A concave upward proton inventory suggests that more than one proton is transferred in a single transition state, likely a conformation change required to open the site and release products. Two groups are involved in the acid-base chemistry of the reaction. One of these groups catalyzes the steps involved in forming the imine between the alpha-amine of glutamate and the aldehyde of AASA. The second group, which has a pKa of 5.6, accepts a proton from the alpha-amine of glutamate so that it can act as a nucleophile in forming a carbinolamine upon attack of the carbonyl of AASA.;Site directed mutagenesis has been carried out to elucidate the role of D125, C154 and Y99 residues in the reaction catalyzed by SR. Kinetic parameters for single and double mutants show a sharp decrease in k cat for the mutants suggesting that these residues are important for the reaction. pH-rate profiles for D125A, C154S, Y99F, D125A-Y99F and D125A-C154S mutants indicate that these residues do not play the role of general acid-base catalyst in the reaction and further studies are required to determine the identity of the general acid-base catalyst. A possible candidate for the general acid-base catalyst is the primary amine of saccharopine and chemical modification needs to be carried out to determine its role in the reaction.;Substrate specificity studies have been carried out using analogues of saccharopine and nicotinamide adenine dinucleotide. Alternative substrates have been used for NADPH in the forward reaction direction at pH 7.0. Inhibition studies using analogues of the dinucleotide show that most of the binding energy comes from the ADP of NADP. Replacement of the carbonyl group of nicotinamide ring with a thio group results in poor binding indicating that the bulky and considerably less electronegative sulphur results in steric hindrance and prevention of hydrogen bonding in the active site of SR, as shown by the high appKm value. The 2' phosphate group plays a significant role in binding which is indicated by the sharp decrease in the V/K value when NADH is employed as an alternative substrate in the forward reaction direction. Dead end analogues of saccharopine suggest that the carboxylate groups need to be flexible and orientation of the carboxylate groups is crucial to binding of saccharopine to SR. (Abstract shortened by UMI.)...