The bifunctional aminoadipic semialdehyde synthase in lysine degradation. Separation of reductase and dehydrogenase domains by limited proteolysis and column chromatography.
The mammalian aminoadipic semialdehyde synthase is a bifunctional enzyme that catalyzes the first two sequential steps in lysine degradation in the major saccharopine pathway (Markovitz, P. J., Chuang, D. T., and Cox, R. P. (1984) J. Biol. Chem. 259, 11643-11646). We show here that limited proteolysis of the highly purified synthase from bovine liver with elastase, chymotrypsin, and papain resulted in separation of lysine-ketoglutarate reductase and saccharopine dehydrogenase activities as judged by activity stainings of the polyacrylamide gel. Enzyme assays showed no loss of the two activities after digestions with these proteases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis disclosed the presence of two limit polypeptides in the elastolytic digests, i.e. fragment A (Mr = 62,700) and fragment B (Mr = 49,200). These fragments were apparently derived from the same polypeptide (Mr = 115,000) of the parent synthase. The reductase and dehydrogenase activities of the elastase-digested synthase were completely resolved by DEAE-Bio-Gel column chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that fragment A and fragment B were associated with reductase and dehydrogenase activities, respectively. The bovine synthase showed Mr = 420,000 in sedimentation equilibrium, confirming a tetrameric structure for the enzyme. The above results establish that the reductase and dehydrogenase domains of the aminoadipic semialdehyde synthase are separately folded and functionally independent of each other.
D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D012443:Saccharopine Dehydrogenases / Q000378:metabolism*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors; C043044:AASS protein, human; D012443:Saccharopine Dehydrogenases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D010206:Papain; D008239:Lysine