Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus.
The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60 degrees C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (KD equal to 2 and 0.5 microm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.
Jensen
Kaj F
KF
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Denmark. kfj@mermaid.molbio.ku.dk
Arent
Susan
S
Larsen
Sine
S
Schack
Lise
L
D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase