Integrated allosteric regulation in the S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein.
BACKGROUND
The S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied.


RESULTS
The results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate.


CONCLUSIONS
This investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated.
Serre
Valérie
V
Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, FRE 2621 CNRS and Université Pierre et Marie Curie, 96 Bd Raspail 75006 Paris, France. serre@ccr.jussieu.fr
Penverne
Bernadette
B
Souciet
Jean-Luc
JL
Potier
Serge
S
Guy
Hedeel
H
Evans
David
D
Vicart
Patrick
P
Hervé
Guy
G
D000494:Allosteric Regulation / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000502:physiology*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D020134:Catalytic Domain / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology*; D009154:Mutation / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D016415:Sequence Alignment / Q000379:methods; D017386:Sequence Homology, Amino Acid; D014544:Uridine Triphosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C019820:carbamoyl phosphate synthase-aspartatecarbamoyl transferase complex; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D014544:Uridine Triphosphate