Comparison of the essential arginine residue in Escherichia coli ornithine and aspartate transcarbamylases

Abstract

The reaction of phenylglyoxal with Escherichia coli ornithine transcarbamylase (carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3) leads to complete loss of enzymatic activity. The behavior of this reagent towards ornithine transcarbamylase is remarkably similar to that observed with E. coli aspartate transcarbamylase (carbamoylphosphate: l-aspartate carbamoyltransferase, EC 2.1.3.2) and its catalytic subunit (Kantrowitz, E.R. and Lipscomb, W.N. (1976) J. Biol. Chem. 251, 2688–2695). The rate of phenylglyoxal inactivation increases in the order ornithine transcarbamylase, catalytic subunit of aspartate transcarbamylase and aspartate transcarbamylase. For ornithine transcarbamylase, the substrate carbamyl phosphate alone or in combination with the substrate analog norvaline protect the enzyme from phenylglyoxal inactivation. Under similar conditions, carbamyl phosphate or carbamyl phosphate plus succinate will protect the catalytic subunit of aspartate transcarbamylase in an almost identical manner. Using [¹⁴C]phenylglyoxal, the number of arginine residues involved in loss of activity was determined to be approx. three per ornithine transcarbamylase molecule or one arginine per active site. The data suggest that the arginine necessary for activity is involved in the binding of carbamyl phosphate to the enzyme. The similarity in phenylglyoxal reactivities combined with genetic and structural data suggest very strongly that there is an evolutionary relationship between ornithine transcarbamylase and the catalytic subunit of aspartate transcarbamylase.