The inactivation of saccharopine dehydrogenase (L-lysine-forming) by diethyl pyrocarbonate.
Saccharopine dehydrogenase (epsilon-N-(L-glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine-forming) EC 1.5.1.7) from baker's yeast is inactivated by diethyl pyrocarbonate. Spectrophotometric studies show that the inactivation results from the modification of 3 histidyl residues/molecule of enzyme. The sulfhydryl content of the enzyme is unchanged by modification. The reversibility of inactivation by hydroxylamine and the pH dependence of inactivation are also consistent with the inactivation being due to modification of the histidyl residue. Although the coenzyme and substrates are without effect when added singly, the inactivation is completely protected by alpha-ketoglutarate in the presence of a saturating concentration of NADH. Since alpha-ketoglutarate binds only to the enzyme . NADH complex, the results suggest that the inactivation is due to modification of the residue at or near the substrate-binding site. Under the conditions where the inactivation is largely protected by NADH plus alpha-ketoglutarate, 2 histidyl residues appear to be modified suggesting that only 1 residue involved in the catalytic activity. The modification appears to prevent the binding of alpha-ketoglutarate, but not of the coenzyme, to the enzyme. The protein fluorescence of the native and modified enzymes is quenched by NAD+ and NADH. However, the NADH titration curve of the modified enzyme is not affected by alpha-ketoglutarate, in contrast to the native enzyme which shows an increase in the apparent affinity for the coenzyme in the presence of alpha-ketoglutarate.
D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000032:analysis; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012443:Saccharopine Dehydrogenases / Q000037:antagonists & inhibitors*; D013056:Spectrophotometry, Ultraviolet
D005561:Formates; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases; D004047:Diethyl Pyrocarbonate