| Saccharopine dehydrogenase (SDH) catalyses the NAD-dependent oxidative deamination of saccharopine to give L-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations of residues singly and in pairs are being made. In this dissertation, the effects of mutation of active site residues, E78, E122, K99 and D319, on reactant binding and catalysis is reported. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, E78A/E122A mutant enzymes in the wild type back ground, and K99M and D319A in the C205S background. Mutations of glutamates 78, 122 and D319, increase the positive charge while mutation on K99, decreases the positive charge in the active site, and as a result the pKa values of the catalytic groups were affected. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E·NADH. Large changes in V/KLys, but not V, suggest that E78, E122, K99 contribute binding energy for lysine. Shifts in the pKa values of the catalytic groups by more than a pH unit to higher and lower pH was observed for the V/KLys pH-rate profile of the glutamate mutant enzymes suggesting that E78 and E122 modulate the basicity of the catalytic groups. Changing K99 affected several kinetic parameters and this is consistent with its contribution to a hydrogen-bonding network in the active site. The largest change in V/KLys was observed for K99M which suggests it is crucial for lysine binding. Substantial changes observed for the second order rate constants V/KNADH and V/K alpha-Kg, consistent with its contribution to a hydrogen bonding network in the active site. Mutation of D319 exhibited its largest effect on V/KNADH and KNADH, suggesting its contribution to binding NADH. No significant changes in V/Et was observed for K99 and D319 indicating they are not catalytic groups but are interactively involved in substrate binding. All ionizable residues studied thus far appear to be important for the reaction and most of them play more than one role. |
