Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis.

The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme. In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme. On the basis of the three-dimensional structure of the enzyme with CTP bound, three residues were selected. Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides. For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme. Furthermore, CTP is able to inhibit the His-20r----Ala enzyme 40% more than the wild-type enzyme. In the case of the Tyr-89r----Ala enzyme. ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70%. These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP. The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS)