Grabner Gail K, Switzer Robert L
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 2003 Feb 28;278(9):6921-7. doi: 10.1074/jbc.M211111200. Epub 2002 Dec 13.
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.
来自枯草芽孢杆菌及许多其他细菌的PyrR蛋白是一种双功能蛋白。其主要功能是通过以尿苷核苷酸依赖的方式与pyr mRNA上的特定位点结合，并改变下游RNA的折叠以促进转录终止，从而调节嘧啶生物合成（pyr）基因的表达。尽管PyrR与其他细菌尿嘧啶磷酸核糖转移酶（UPRTase）的氨基酸序列相似度很低，但它也催化尿嘧啶磷酸核糖转移酶反应。在所有检测条件下，PyrR催化的UPRTase反应均遵循乒乓稳态动力学模式，但未检测到[(14)C]尿嘧啶-UMP和[(32)P]PP(i)-磷酸核糖焦磷酸交换反应的催化作用。已证明，对于PyrR的有序双双反应机制的稳态方程，包括在PRPP结合后但尿嘧啶结合前发生的动力学不可逆构象变化，可解释该酶的乒乓模式。以下实验观察结果支持了这一机制。逆反应极其缓慢，催化速率常数比正反应小3300倍。正反应的产物抑制模式与不可逆构象变化模型的一个版本一致，在该模型中，PyrR在UMP解离之前恢复到未结合配体的构象，并且与其他几种动力学机制不一致。通过平衡透析表明，UMP和磷酸核糖焦磷酸与游离的PyrR结合（解离常数分别为27±3和18±2微摩尔），但在高达750微摩尔的平衡浓度下，尿嘧啶和PP(i)不结合。我们提出，为PyrR建立的构象变化动力学模型也可以解释许多其他关于各种磷酸核糖转移酶的乒乓动力学的报道，这些酶不会形成经典乒乓动力学预测的磷酸核糖-酶中间体。
