Tao W, Grubmeyer C, Blanchard J S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1996 Jan 9;35(1):14-21. doi: 10.1021/bi951898l.
Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate to orotidine 5'-monophosphate (OMP) and pyrophosphate. We have determined kinetic isotope effects on the reaction of OMP with pyrophosphate and with the pyrophosphate analog phosphonoacetic acid. In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosphorylytic reaction. The transition state resembles a classical oxocarbonium ion. Using the recently reported three-dimensional structures of the OPRTase-OMP (Scapin et al., 1994) and the OPRTase-PRPP complexes (Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase. We propose a detailed chemical mechanism which is consistent with these results.
乳清酸磷酸核糖基转移酶（OPRTase）催化α-D-磷酸核糖焦磷酸（PRPP）和乳清酸在镁离子依赖下转化为乳清苷5'-单磷酸（OMP）和焦磷酸。我们已经测定了OMP与焦磷酸以及焦磷酸类似物膦酰乙酸反应的动力学同位素效应。在后一种情况下，动力学同位素效应的充分表达使我们能够计算焦磷酸解反应的过渡态结构。过渡态类似于经典的氧鎓离子。利用最近报道的OPRTase - OMP（斯卡平等人，1994年）和OPRTase - PRPP复合物（斯卡平等人，1995年a）的三维结构，我们将计算出的过渡态结构模拟到OPRTase的活性位点中。我们提出了一个与这些结果一致的详细化学机制。
