The conserved residues glutamate-37, aspartate-100, and arginine-269 are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. The X-ray structure of this enzyme indicates that the side chains of His-41, Asp-100, and Asp-90 from one catalytic chain form interactions with the side chains of Glu-37, Arg-65, and Arg-269, respectively, from an adjacent catalytic chain. In order to determine whether these interactions are important for the structural stabilization of the enzyme and/or homotropic and heterotropic effects, four mutant versions of aspartate transcarbamoylase, Glu-37-->Ala, Asp-100-->Asn, Asp-100-->Ala, and Arg-269-->Ala, were created by site-specific mutagenesis. The Glu-37-->Ala holoenzyme exhibits essentially wild-type behavior with respect to homotropic cooperativity and heterotropic regulation by ATP and CTP. The Glu-37-->Ala catalytic subunit exhibits a half-life of inactivation at 69 +/- 0.5 degrees C of 4.9 min, as compared to 5.8 min for the wild-type catalytic subunit. The Asp-100-->Asn and Asp-100-->Ala holoenzymes are slightly more active than the wild-type holoenzyme, exhibit 1.4-fold and 1.8-fold reductions in the aspartate concentration at half the maximal specific activity, respectively, and show increased affinities for ATP and CTP. Both the Asp-100-->Asn and Asp-100--> Ala catalytic subunits exhibit a 2-fold reduction in the half-life of inactivation at 69 +/- 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)