title: Chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae.
abstract: 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 imine, 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
authors.forename: Ashwani Kumar
authors.initials: AK
authors.lastname: Vashishtha
authors.forename: Ann H
authors.initials: AH
authors.lastname: West
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D041961:Deuterium Exchange Measurement; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D012443:Saccharopine Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D009249:NADP; D012443:Saccharopine Dehydrogenases; C041897:saccharopine dehydrogenase (NADP, L-glutamate-forming); D008239:Lysine; C100169:saccharopine