title: Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain.
abstract: The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site.
authors.affiliation: Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR 7631 CNRS-Université Pierre et Marie Curie, 96 Bd Raspail 75006 Paris, France.
authors.forename: V
authors.initials: V
authors.lastname: Serre
authors.forename: H
authors.initials: H
authors.lastname: Guy
authors.forename: B
authors.initials: B
authors.lastname: Penverne
authors.forename: M
authors.initials: M
authors.lastname: Lux
authors.forename: A
authors.initials: A
authors.lastname: Rotgeri
authors.forename: D
authors.initials: D
authors.lastname: Evans
authors.forename: G
authors.initials: G
authors.lastname: Hervé
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000502:physiology; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology; D002221:Carbamyl Phosphate / Q000378:metabolism*; D005246:Feedback; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000502:physiology; D010957:Plasmids; D011743:Pyrimidines / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D009097:Multienzyme Complexes; D011743:Pyrimidines; C019820:carbamoyl phosphate synthase-aspartatecarbamoyl transferase complex; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid; D014544:Uridine Triphosphate