mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila

• Published in: Journal of molecular biology Vol. 287; no. 2; pp. 277 - 285
• Main Authors: Simmons, A.J, Rawls, J.M, Piskur, J, Davidson, J.N
• Format: Journal Article
• Language: English
• Published: England 26-03-1999
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; Allosteric Regulation - genetics; Animals; Animals, Genetically Modified; binding sites; Binding Sites - genetics; Carbamoyl-Phosphate Synthase (Ammonia); Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - genetics; Drosophila; Drosophila - enzymology; Drosophila - genetics; Drosophila melanogaster; Drosophila Proteins; enzyme activity; Feedback; inhibition; Insect Proteins - genetics; Kinetics; ligases; magnesium; Magnesium - pharmacology; molecular conformation; Multienzyme Complexes - genetics; mutation; Mutation, Missense - genetics; Phenotype; pyrimidine nucleotides; regulation; Sequence Alignment; Sequence Analysis, DNA; transgenic animals; Uridine Triphosphate - pharmacology
• DOI: 10.1006/jmbi.1999.2618
• EISSN: 1089-8638
• ISSN: 0022-2836

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.