title: Contribution of K99 and D319 to substrate binding and catalysis in the saccharopine dehydrogenase reaction.
abstract: Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to l-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V(2)/K(m) values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/K(Lys) increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V(2)/K(NADH) by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, United States.
authors.forename: Devi K
authors.initials: DK
authors.lastname: Ekanayake
authors.forename: Ann H
authors.initials: AH
authors.lastname: West
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D007097:Imines / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012443:Saccharopine Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D007097:Imines; D007656:Ketoglutaric Acids; D009243:NAD; D012443:Saccharopine Dehydrogenases; D008239:Lysine