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. The Arg-269 --> Ala holoenzyme exhibits a 2-fold reduction in the maximal specific activity, normal homotropic cooperativity, but 12-fold and 44-fold increases in the aspartate and carbamoyl phosphate concentrations at half the maximal specific activity, respectively. Furthermore, the Arg-269 --> Ala enzyme shows a 1.4-fold and 17-fold reduction in the affinity for ATP and CTP, respectively, as well as a 40-fold reduction in the maximal activation by ATP and a 3-fold reduction in the maximal inhibition by CTP. Replacement of Arg-269 by Ala also results in a dramatic loss of stability. At 50.25 +/- 0.25-degrees-C, a temperature at which the wild-type holoenzyme is stable, the Arg-269 --> Ala holoenzyme is inactivated completely with a half-life of 2.0 min.