Chemical Mechanism of Saccharopine Reductase from Saccharomyces cerevisiae

Saccharopine reductase (SR) [saccharopine dehydrogenase (L-glutamate forming), EC 1.5.1.10] catalyzes the condensation of L-alpha-aminoadipate-delta-semialdehyde (AASA) with L-glutamate to give an]mine, which is reduced by NADPH to give saccharopine. 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 group, which has a pK(a) of about 8, is observed in the pH-rate profiles for V-1 and V-1/K-Glu and must be protonated for optimal activity. It is also observed in the V-2 and V-2/K-Sacc pH-rate profiles and is required unprotonated. The second group, which has a pK(a) 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.