Irreversible inactivation of aspartate aminotransferase by 2-oxoglutaconic acid and its dimethyl ester.
Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25 degrees C (pH 7.0) results in slight inactivation (approximately 15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and alpha-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40 mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t 1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40 mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10 mM L-glutamate, pH 7.2, 25 degrees C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25 degrees C, pH 7.2) results in significant inactivation (approximately 30%). The enzyme is protected against inactivation by the presence of alpha-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1 approximately 2 mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate approximately 0.01 min-1; pH 6.0, 25 degrees C; t 1/2 max approximately 70 min). This finding suggests that 2-oxoglutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated alpha-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with alpha-ketoglutarate. The results suggest that unsaturated alpha-keto acids and their esters may be useful probes for the study of alpha-keto acid-utilizing enzymes.
Biotechnology Research Center, Toyama Prefectural University.
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Kato
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Asano
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Makar
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Cooper
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D013552:Swine
D004791:Enzyme Inhibitors; D007656:Ketoglutaric Acids; C104056:dimethyl 2-oxoglutaconate; C035553:3,4-didehydro-2-ketoglutaric acid; D001219:Aspartate Aminotransferases; D003545:Cysteine