Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.