THE USE OF ALANINE SCANNING MUTAGENESIS TO DETERMINE THE ROLE OF THE N-TERMINUS OF THE REGULATORY CHAIN IN THE HETEROTROPIC MECHANISM OF ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE

The location of the first seven residues of the regulatory chain of Escherichia coli aspartate transcarbamoylase has been identified by X-ray crystallography to be near the binding site of the regulatory nucleotides. In order to determine the function of the N-terminus of the regulatory chain of aspartate transcarbamoylase in heterotropic regulation, alanine scanning mutagenesis was used. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7r were each replaced with alanine. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, three of the mutant enzymes, Asp4r --> Ala, Lys6r --> Ala and Leu7r --> Ala, exhibited notable changes in their response to the regulatory nucleotides, while mutations at Thr2r, His3r and Asn5r exhibited only minor changes in their heterotropic responses. For the Asp4r --> Ala enzyme, the responses to ATP and CTP were reduced similar to 30 and 40% respectively, compared with the wild-type enzyme. For the Lys6r --> Ala enzyme, the response to ATP was reduced similar to 70%, while the CTP response was reduced similar to 50%. In the case of the Leu7r --> Ala enzyme, a 30 and 20% reduction in response to ATP and CTP respectively, was observed, The synergistic inhibition by UTP in the presence of CTP for the Lys6r --> Ala enzyme was reduced similar to 40% compared with that of the wild type enzyme. For the Leu7r --> Ala enzyme, the synergistic inhibition was abolished. In addition, UTP decreased the CTP binding affinity of the Leu7r --> Ala enzyme. Analysis of the kinetic data from these mutant enzymes suggests that residues Thr2r, His3r and Asn5r have little effect on the heterotropic mechanism, while residues Asp4r, Lys6r and Leu7r play a more significant role in the heterotropic response of the enzyme toward the nucleotides. Furthermore, residue Leu7r appears to be directly involved in the mechanism for synergistic inhibition of aspartate transcarbamoylase. In this study alanine scanning mutagenesis has provided a rapid method of identifying those residues in the N-terminal region of the regulatory chain of aspartate transcarbamoylase important for heterotropic regulation.