Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation.
A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.
D000818:Animals; D002950:Citrate (si)-Synthase / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D004794:Enzyme Repression; D005649:Fumarate Hydratase / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008024:Ligands / Q000494:pharmacology*; D008190:Lyases / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010770:Phosphotransferases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D013696:Temperature; D013769:Tetrahymena pyriformis / Q000201:enzymology*
D008024:Ligands; D008623:Mercaptoethanol; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D003605:D-Amino-Acid Oxidase; D002950:Citrate (si)-Synthase; D010770:Phosphotransferases; D006593:Hexokinase; D011770:Pyruvate Kinase; D010447:Peptide Hydrolases; D008190:Lyases; D005649:Fumarate Hydratase