Identification of the functionally important cysteinyl residue in pig heart aspartate aminotransferase

L-Aspartate:2-oxoglutarate aminotransferase (EC 2.6.1.1) from pig heart cytosol consists of two identical subunits of molecular weight about 46,500 [l]. Each subunit has 5 thiol groups [2-51: two exposed SH groups readily modified by iodoacetic acid with no decrease of activity, two fully buried SH groups inaccessible to thiol reagents in the native enzyme, and one relatively non-reactive, functionally important SH group, which can be selectively blocked by p-chloro-mercuribenzoate @CMB), with 95% inactivation of the enzyme, after previous alkylation of the two exposed SH groups. It has been shown that selective mercaptidation of this SH group leads to considerable impairment of the enzyme’s affinity for substrate analogues [5]. From the recent findings of Birchmeier and Christen [6] it appears that aminotransferase is similarly 95% inactivated when the functionally important SH group is subjected to “syncatalytic” alkylation with N-ethylmaleimide in the presence of a substrate pair. Modification of the functional SH group may be of critical significance for activity as a consequence of its proximity to the active site. We have achieved selective labeling of this group by syncatalytic alkylation with N-ethyl[14C]maleimide in the presence of glutamate and cu-ketoglutarate, We have also carried out alkylation of partly and fully buried SH groups of the enzyme with iodo[‘4C]acetic acid in 8 M urea and isolated radioactive tryptic peptides; one of these proved identical (in amino acid composition and nature of the N-terminal residue) with the labeled peptide isolated after syncatalytic alkylation of the enzyme with N-ethyl [14C]maleimide. All experiments were per-