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 chainof Escherichia coli aspartate transcarbamoylase has been identifiedby X-ray crystallography to be near the binding site of theregulatory nucleotides. In order to determine the function ofthe N-terminus of the regulatory chain of aspartate transcarbamoylasein heterotropic regulation, alanine scanning mutagenesis wasused. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7rwere each replaced with alanine. Analyses of these mutant enzymesindicate that none of these substitutions significantly alterthe catalytic properties of the enzyme. However, three of themutant enzymes, Asp4r Ala, Lys6r Ala and Leu7r Ala, exhibitednotable changes in their response to the regulatory nucleotides,while mutations at Thr2r, His3r and Asn5r exhibited only minorchanges in their heterotropic responses. For the Asp4r Alaenzyme, the responses to ATP and CTP were reduced 30 and 40%respectively, compared with the wild-type enzyme. For the Lys6r Ala enzyme, the response to ATP was reduced 70%, while theCTP response was reduced 50%. In the case of the Leu7r Alaenzyme, a 30 and 20% reduction in response to ATP and CTP respectively,was observed. The synergistk inhibition by UTP in the presenceof CTP for the Lys6r Ala enzyme was reduced 40% compared withthat of the wild type enzyme. For the Leu7r Ala enzyme, thesynergistic inhibition was abolished. In addition, UTP decreasedthe CTP binding affinity of the Leu7r Ala enzyme. Analysisof the kinetic data from these mutant enzymes suggests thatresidues Thr2r, His3r and Asn5r have little effect on the heterotropicmechanism, while residues Asp4r, Lys6r and Leu7r play a moresignificant role in the heterotropic response of the enzymetoward the nucleotides. Furthermore, residue Leu7r appears tobe directly involved in the mechanism for synergistic inhibitionof aspartate transcarbamoylase. In this study alanine scanningmutagenesis has provided a rapid method of identifying thoseresidues in the N-terminal region of the regulatory chain ofaspartate transcarbamoylase important for heterotropic regulation.