Kinetic studies of the uracil phosphoribosyltransferase reaction catalyzed by the Bacillus subtilis pyrimidine attenuation regulatory protein PyrR.

The PyrR protein from Bacillus subtilis and many other bacteria is a bifunctional protein. Its primary function is the regulation of expression of pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA in a uridine nucleotide-dependent manner and altering the folding of downstream RNA to promote termination of transcription. PyrR also catalyzes the uracil phosphoribosyltransferase (UPRTase) reaction even though it bears little amino acid sequence similarity to other bacterial UPRTases. The PyrR-catalyzed UPRTase reaction obeyed a Ping Pong steady state kinetic pattern under all conditions examined, but no catalysis of [(14)C]uracil-UMP and [(32)P]PP(i)-phosphoribosylpyrophosphate exchange reactions could be detected. Steady state equations for Ordered Bi Bi mechanisms for PyrR that include a kinetically irreversible conformational change after binding of PRPP but before uracil binding were shown to account for the Ping Pong pattern of the enzyme. This mechanism was supported by the following experimental observations. The reverse reaction was extremely slow with a catalytic rate constant 3300 times smaller than for the forward reaction. Patterns of product inhibition of the forward reaction were consistent with a version of the irreversible conformational change model in which PyrR returns to the unliganded conformation before dissociation of UMP and were inconsistent with several other kinetic mechanisms. UMP and phosphoribosylpyrophosphate were shown by equilibrium dialysis to bind to free PyrR (dissociation constants of 27 +/- 3 and 18 +/- 2 microm, respectively), but uracil and PP(i) did not bind at equilibrium concentrations up to 750 microm. We propose that the conformational change kinetic model developed for PyrR can also account for numerous other reports of Ping Pong kinetics for various phosphoribosyltransferases that do not form the phosphoribosyl-enzyme intermediate predicted by classic Ping Pong kinetics.