Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate β-lyases, but turnover leads to inactivation

Abstract

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT m and BCAT c) are homodimers in the fold type IV class of pyridoxal 5′-phosphate-containing enzymes that also contains d-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a β-lyase). Recombinant human BCAT m and BCAT c were shown to have β-lyase activity toward three toxic cysteine S-conjugates [ S-(1,1,2,2-tetrafluoroethyl)- l-cysteine, S-(1,2-dichlorovinyl)- l-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)- l-cysteine] and toward β-chloro- l-alanine. Human BCAT m is a much more effective β-chloro- l-alanine β-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT m, but not BCAT c, also exhibits measurable β-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl- l-cysteine]. Benzothiazolyl- l-cysteine, however, inhibits the l-leucine–α-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT m. In the presence of β-lyase substrates and α-ketoisocaproate (the α-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the β position, β-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the β-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is ∼170–280 for BCAT m and ∼40–50 for BCAT c. Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)- l-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.