Purification and characterization of saccharopine dehydrogenase from baker's yeast.
Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity. The overall purification was about 1,200-fold over the crude extract with a yield of about 24%. The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S. The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme. A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography. The enzyme had a high isoelectric pH of 10.1. The NH2-terminal sequence was Ala-Ala----. The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present. Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity. Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.
D000596:Amino Acids / Q000032:analysis; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012443:Saccharopine Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases