Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-
a-1-diphosphate (PRPP) and uracil to uridine monophosphate
(UMP) and diphosphate (PPi). The tetrameric enzyme from Sulfolobus
solfataricus has a unique type of allosteric regulation by cytidine
triphosphate (CTP) and guanosine triphosphate (GTP). Here we report
two structures of the activated state in complex with GTP. One structure
(refined at 2.8-Å resolution) contains PRPP in all active sites, while the other
structure (refined at 2.9-Å resolution) has PRPP in two sites and the
hydrolysis products, ribose-5-phosphate and PPi, in the other sites.
Combined with three existing structures of uracil phosphoribosyltransferase
in complex with UMP and the allosteric inhibitor cytidine triphosphate
(CTP), these structures provide valuable insight into the mechanism of
allosteric transition from inhibited to active enzyme. The regulatory triphosphates
bind at a site in the center of the tetramer in a different manner
and change the quaternary arrangement. Both effectors contact Pro94 at the
beginning of a long ß-strand in the dimer interface, which extends into a
flexible loop over the active site. In the GTP-bound state, two flexible loop
residues, Tyr123 and Lys125, bind the PPi moiety of PRPP in the neighboring
subunit and contribute to catalysis, while in the inhibited state, they
contribute to the configuration of the active site for UMP rather than PRPP
binding. The C-terminal Gly216 participates in a hydrogen-bond network in
the dimer interface that stabilizes the inhibited, but not the activated, state.
Tagging the C-terminus with additional amino acids generates an
endogenously activated enzyme that binds GTP without effects on activity.