Modification of three active site lysine residues in the catalytic subunit of aspartate transcarbamylase by D- and L-bromosuccinate.

Abstract

Treatment of the catalytic subunit of aspartate transcarbamylase from Escherichia coli with either D- or Lbromosuccinate at pH 8.5 results in a loss of catalytic activity. Succinate, an analog of the substrate L-aspartate, affords some protection against inactivation, while the putative transition state analogN-(phosphonacetyl)-L-aspartate provides complete protection. The substrate carbamyl phosphate provides greater protection against inactivation by L-bromosuccinate than by D-bromosuccinate. Complete loss of activity is accompanied by incorporation of approximately 1.3 succinate moieties per catalytic chain resulting from partial modification of 3 lysine residues, identified as numbers 83, 84, and 224 in the preliminary catalytic chain sequence of W. Konigsberg (personal communication). A significant number of catalytic chains are modified at both positions 83 and 84. In the absence of ligands, D-bromosuccinate reacts with lysine 83 to a greater extent than does the L isomer. Bulky inhibitors such as CTP and pyridoxal 5’-phosphate provide varying degrees of protection against inactivation and overall modification without altering significantly the relative extent of alkylation of the 3 residues. However, carbamyl phosphate not only protects against inactivation and overall modification, but also selectively suppresses alkylation of lysine 83 and eliminates the production of catalytic chains modified at both lysines 83 and 84. The results suggest that all 3 lysine residues are at or near the active site, that modification of any one of them causes loss of catalytic activity, and that lysine 83 is at or near the carbamyl phosphate binding site.