| Lysine is an essential amino acid in mammals including humans. The alpha-aminoadipate (AAA) pathway for lysine biosynthesis is unique to fungal organisms and enzymes of this pathway are of interest as potential targets for anti-fungal drug design. Saccharopine dehydrogenase (SDH) [N6-(glutaryl-2)-L-lysine: NAD-oxidoreductase (L-lysine-forming) (EC 1.5.1.7)] catalyzes the reversible NAD-dependent oxidative deamination of saccharopine to generate L-lysine and alpha-ketoglutarate. A disulfide bond between cysteines 205 and 249 is observed in structures of the apoenzyme and those with sulfate or oxalylglycine bound to the substrate-binding domain. However, in the structure with adenosine monophosphate bound in the dinucleotide-binding site, the disulfide bond is reduced.;In this dissertation, site-directed mutagenesis was used to generate the C205S mutant enzyme, which was characterized kinetically using a combination of steady state kinetics, pH-rate profiles, and isotope effects. The C205S mutant enzyme was also characterized crystallographically.;The kinetic and chemical mechanisms of the C205S mutant enzyme remained unchanged compared to those of the wild type enzyme. At pH 7, the first ( V/Et) and second order (V/K Et) rate constants increased for the C205S mutant enzyme compared to wild type. Differences in the pH-rate profiles for V1, V1/KNAD, V2, and V2/Kalpha-Kg compared to wild type indicated the increase in activity resulted from a decrease in the sensitivity of the C205S mutant enzyme to the protonation state of a group responsible for a pH-dependent conformational change. The increased rate of the C205S mutant enzyme was also reflected in larger deuterium and solvent deuterium isotope effects on V2 and V2/ KLys, with no significant change in the multiple isotope effect in which the solvent isotope effect was repeated with NADD. Thus, the rate of central complex interconversion contributes more to rate limitation, but the ratio of the rates of the hydride transfer step and imine hydrolysis is the same. Overall, data suggested the wild type enzyme is a mixture of disulfide- and dithiol-containing species, with the former dominating.;Structures of the C205S apoenzyme and one with NADH bound in the dinucleotide binding site have been solved. Structural data suggested the disulfide between the C205 and C249 in the wild type enzyme might prevent the dinucleotide binding. Movement of arginine 204, 130, and 131 side chains upon dinucleotide binding and the resulting positioning of arginine 131 into the correct location for the substrate binding has been proposed as a structural basis for the ordered kinetic mechanism proposed previously for SDH. Also, a role for the network of electrostatic interactions between K13, K77, H96, E16, and E78 has been proposed in positioning K77 and H96 for reaction and modulating the p Kas of these catalytic side chains close to neutrality, with K13 playing an essential part.;To probe the acid-base chemical mechanism of saccharopine dehydrogenase, K77M and H96Q, K77M/C205S and H96Q/C205S, and K77M/H96Q/C205S single, double, and triple mutants, respectively, were prepared and characterized via initial velocity studies and isotope effects. A 100-fold decrease in kcat and a large D(V/KLys) isotope effect suggest K77 is the base that accepts a proton from the secondary amine of saccharopine. A 4-fold decrease in kcat and a large D 2O(V/KLys) isotope effect suggest H96 is the base that catalyzes the imine hydrolysis via the activation of a water molecule. |
