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 approximately 30 and 40% respectively, compared with the wild-type enzyme. For the Lys6r-->Ala enzyme, the response to ATP was reduced approximately 70%, while the CTP response was reduced approximately 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 approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Dembowski
N J
NJ
Merkert Chemistry Center, Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
Kantrowitz
E R
ER
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D000409:Alanine