title: Supporting role of lysine 13 and glutamate 16 in the acid-base mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.
abstract: Saccharopine dehydrogenase (SDH) catalyzes the NAD+ dependent oxidative deamination of saccharopine to form lysine (Lys) and α-ketoglutarate (α-kg). The active site of SDH has a number of conserved residues that are believed important to the overall reaction. Lysine 13, positioned near the active site base (K77), forms a hydrogen bond to E78 neutralizing it, and contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate 16 is within hydrogen bond distance to the Nε atom of R18, which has strong H-bonding interactions with the α-carboxylate and α-oxo groups of α-kg. Mutation of K13 to M and E16 to Q decreased kcat by about 15-fold, and primary and solvent deuterium kinetic isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting for the overall reaction. The pH-rate profiles for K13M exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pKas of catalytic groups. Elimination of E16 affects optimal positioning of R18, which is involved in binding and holding α-kg in the correct conformation for optimum catalysis. In agreement, a ΔΔG°' of 2.60 kcal/mol is estimated from the change in Kα-kg for replacing E16 with Q.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States.
authors.forename: Vidya Prasanna
authors.initials: VP
authors.lastname: Kumar
authors.forename: Ann H
authors.initials: AH
authors.lastname: West
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D000136:Acid-Base Equilibrium; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012443:Saccharopine Dehydrogenases / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018698:Glutamic Acid; D012443:Saccharopine Dehydrogenases; D008239:Lysine