caa a22 4500 397556470 CHVBK 20180308164803.0 cr unu---uuuuu 161202e199609 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021446 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021446 Irreversible Inactivation of Aspartate Aminotransferase by 2-Oxoglutaconic Acid and Its Dimethyl Ester [Elektronische Daten] [Yasuo Kato, Yasuhisa Asano, Tapas Makar, Arthur J.L. Cooper] Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5′-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25° (pH 7.0) results in slight inactivation (˜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-oxoglut.aconic acid dimethyl ester is most pronounced in the presence of both glutamate and a-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 I 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 t1/2 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°C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25°C, pH 7.2) results in significant inactivation (˜30%). The enzyme is protected against inactivation by the presence of α-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 kineties (K1˜2mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L glutamate ˜0.01 min−1; pH 6.0, 25°C; t1/2max ˜70 min). This finding suggests that 2-oxo- glutaconate does not readily react in a syncatslytic 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 α-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 a-ketoglutarate. The results suggest that unsaturated α-keto acids and their esters may be useful probes for the study of α-keto acid-utilizing enzymes. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence aspartate aminotransferase nationallicence glutamate dehydrogenase nationallicence 2-oxoglutaconate nationallicence 2-oxoglutaconic acid dimethylester nationallicence syncatalytic inactivation nationallicence Kato Yasuo Biotechnology Research Center, Toyarna Prefectural University 5180 Kurokawa, Kosugi Toyama 939-03 aut Asano Yasuhisa Biotechnology Research Center, Toyarna Prefectural University 5180 Kurokawa, Kosugi Toyama 939-03 aut Makar Tapas Departments of Biochemistry, Cornell University Medical College New York NY 10021, USA aut Cooper Arthur J.L. Departments of Biochemistry, Cornell University Medical College New York NY 10021, USA aut The Journal of Biochemistry Oxford University Press 120/3(1996-09), 531-539 0021-924X 120:3<531 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021446 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021446 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kato Yasuo Biotechnology Research Center, Toyarna Prefectural University 5180 Kurokawa, Kosugi Toyama 939-03 aut NATIONALLICENCE 700 1- Asano Yasuhisa Biotechnology Research Center, Toyarna Prefectural University 5180 Kurokawa, Kosugi Toyama 939-03 aut NATIONALLICENCE 700 1- Makar Tapas Departments of Biochemistry, Cornell University Medical College New York NY 10021, USA aut NATIONALLICENCE 700 1- Cooper Arthur J.L. Departments of Biochemistry, Cornell University Medical College New York NY 10021, USA aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/3(1996-09), 531-539 0021-924X 120:3<531 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford