title: The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH.
abstract: Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E(t) and V₁/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
authors.forename: Kostyantyn D
authors.initials: KD
authors.lastname: Bobyk
authors.forename: Sang Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Vidya Prasanna
authors.initials: VP
authors.lastname: Kumar
authors.forename: Sung-Kun
authors.initials: SK
authors.lastname: Kim
authors.forename: Ann H
authors.initials: AH
authors.lastname: West
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004271:DNA, Fungal / Q000235:genetics; D041961:Deuterium Exchange Measurement; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012443:Saccharopine Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D001051:Apoenzymes; D004271:DNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D009243:NAD; D012443:Saccharopine Dehydrogenases; D008239:Lysine; D003545:Cysteine; C100169:saccharopine