Chemical mechanism of saccharopine dehydrogenase (NAD+, L-lysine-forming) as deduced from initial rate pH studies.
The variation of kinetic parameters with pH has been determined so as to gain insight into the chemical mechanism of the saccharopine dehydrogenase (NAD+,L-lysine-forming)-catalyzed reaction. In the direction of reductive condensation of lysine and alpha-ketoglutarate (reverse reaction), the V/K profile for lysine shows a group with a pK of 6.3 must be unprotonated and a group with a pK of 8.0 must be protonated for activity. Similar pK's are obtained in the pKi profile for ornithine, which acts as a linear competitive inhibitor with respect to lysine. Temperature and solvent perturbation studies show that these groups are probably histidines. The V/K profile for alpha-ketoglutarate reveals a single group with pK = 8.4 (probably lysine) that must be protonated. It is proposed that one of the histidines is involved in the binding of the epsilon-amino group of the substrate lysine and the positively charged lysine residue hydrogen bonds to the carbonyl oxygen of alpha-ketoglutarate. In the direction of saccharopine cleavage, the V/K profile for saccharopine shows that two groups with pK values of 6.0 and 7.1, possibly a histidine and lysine, must be unprotonated for its reaction with the enzyme X NAD+ complex. The log V-pH plots for the forward and reverse reactions both show sigmoidal curves. At low pH, the activity is lower for the forward reaction, and is higher for the reverse reaction. The ionization of a single group appears to be responsible for the change in activity. A tentative scheme for the chemical reaction is presented.
D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D012443:Saccharopine Dehydrogenases / Q000378:metabolism*
D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases; C029496:saccharopine dehydrogenase (NADP+, lysine-forming)